I-CreI  meganuclease variants with modified specificity, method of preparation and uses thereof

ABSTRACT

Method of preparing I-CreI meganuclease variants having a modified cleavage specificity, variants obtainable by said method and their applications either for cleaving new DNA target or for genetic engineering and genome engineering for non-therapeutic purposes. Nucleic acids encoding said variants, expression cassettes comprising said nucleic acids, vectors comprising said expression cassettes, cells or organisms, plants or animals except humans, transformed by said vectors.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 12/859,905(abandoned), filed Aug. 20, 2010, which is a continuation of U.S. Ser.No. 11/908,798 (now U.S. Pat. No. 7,897,372), filed on Sep. 17, 2007,which is a 35 U.S.C. §371 National Stage patent application ofPCT/IB2006/001203, filed on Mar. 15, 2006, which claims priority under35 U.S.C. §120 and 35 U.S.C. §365(c) to International patent applicationPCT/IB2005/003083, filed on Sep. 19, 2005, and also claims priorityunder 35 U.S.C. §120 and 35 U.S.C. §365(c) to International patentapplication PCT/IB2005/000981, filed Mar. 15, 2005.

BACKGROUND OF THE INVENTION

The present invention relates to a method of preparing I-CreImeganuclease variants having a modified cleavage specificity. Theinvention relates also to the I-CreI meganuclease variants obtainable bysaid method and to their applications either for cleaving new DNA targetor for genetic engineering and genome engineering for non-therapeuticpurposes.

The invention also relates to nucleic acids encoding said variants, toexpression cassettes comprising said nucleic acids, to vectorscomprising said expression cassettes, to cells or organisms, plants oranimals except humans, transformed by said vectors.

Meganucleases are sequence specific endonucleases recognizing large (>12bp; usually 14-40 bp) DNA cleavage sites (Thierry and Dujon, 1992). Inthe wild, meganucleases are essentially represented by homingendonucleases, generally encoded by mobile genetic elements such asinteins and class I introns (Belfort and Roberts, 1997; Chevalier andStoddard, 2001). Homing refers to the mobilization of these elements,which relies on DNA double-strand break (DSB) repair, initiated by theendonuclease activity of the meganuclease. Early studies on the HO(Haber, 1998; Klar et al., 1984; Kostriken et al., 1983), I-SceI(Colleaux et al., 1988; Jacquier and Dujon, 1985; Perrin et al., 1993;Plessis et al., 1992) and I-TevI (Bell-Pedersen et al., 1990;Bell-Pedersen et al., 1989; Bell-Pedersen et al., 1991; Mueller et al.,1996) proteins have illustrated the biology of the homing process. Onanother hand, these studies have also provided a paradigm for the studyof DSB repair in living cells.

General asymmetry of homing endonuclease target sequences contrasts withthe characteristic dyad symmetry of most restriction enzyme recognitionsites. Several homing endonucleases encoded by introns ORF or inteinshave been shown to promote the homing of their respective geneticelements into allelic intronless or inteinless sites. By making asite-specific double-strand break in the intronless or inteinlessalleles, these nucleases create recombinogenic ends, which engage in agene conversion process that duplicates the coding sequence and leads tothe insertion of an intron or an intervening sequence at the DNA level.

Homing endonucleases fall into 4 separated families on the basis ofpretty well conserved amino acids motifs [for review, see Chevalier andStoddard (Nucleic Acids Research, 2001, 29, 3757-3774)]. One of them isthe dodecapeptide family (dodecamer, DOD, D1-D2, LAGLIDADG (SEQ ID NO:91), P1-P2). This is the largest family of proteins clustered by theirmost general conserved sequence motif: one or two copies (vast majority)of a twelve-residue sequence: the dodecapeptide. Homing endonucleaseswith one dodecapeptide (D) are around 20 kDa in molecular mass and actas homodimers. Those with two copies (DD) range from 25 kDa (230 aminoacids) to 50 kDa (HO, 545 amino acids) with 70 to 150 residues betweeneach motif and act as monomer. Cleavage is inside the recognition site,leaving 4 nt staggered cut with 3′H overhangs. Enzymes that contain asingle copy of the LAGLIDADG (SEQ ID NO: 91) motif, such as I-CeuI andI-CreI act as homodimers and recognize a nearly palindromic homing site.

The sequence and the structure of the homing endonuclease I-CreI (pdbaccession code 1g9y) have been determined (Rochaix J D et al., NAR,1985, 13, 975-984; Heath P J et al., Nat. Struct. Biol., 1997, 4,468-476; Wang et al., NAR, 1997, 25, 3767-3776; Jurica et al. Mol. Cell,1998, 2, 469-476) and structural models using X-ray crystallography havebeen generated (Heath et al., 1997).

I-CreI comprises 163 amino acids (pdb accession code 1g9y); saidendonuclease cuts as a dimer. The LAGLIDADG (SEQ ID NO: 91) motifcorresponds to residues 13 to 21; on either side of the LAGLIDADG (SEQID NO: 91) α-helices, a four β-sheet (positions 21-29; 37-48; 66-70 and73-78) provides a DNA binding interface that drives the interaction ofthe protein with the half-site of the target DNA sequence. Thedimerization interface involves the two LAGLIDADG (SEQ ID NO: 91) helixas well as other residues.

The homing site recognized and cleaved by I-CreI is 22-24 bp in lengthand is a degenerate palindrome (see FIG. 2 of Jurica M S et al, 1998 andSEQ ID NO:65). More precisely, said I-CreI homing site is asemi-palindromic 22 bp sequence, with 7 of 11 bp identical in eachhalf-site (Seligman L M et al., NAR, 2002, 30, 3870-3879).

The endonuclease-DNA interface has also been described (see FIG. 4 ofJurica M S et al, 1998) and has led to a number of predictions aboutspecific protein-DNA contacts (Seligman L M et al., Genetics, 1997, 147,1653-1664; Jurica M S et al., 1998; Chevalier B. et al., Biochemistry,2004, 43, 14015-14026).

It emerges from said documents that:

-   -   the residues G19, D20, Q47, R51, K98 and D137 are part of the        endonucleolytic site of I-CreI;    -   homing site sequence must have at least 20 bp to achieve a        maximal binding affinity of 0.2 nM;    -   sequence-specific contacts are distributed across the entire        length of the homing site;    -   base-pair substitutions can be tolerated at many different        homing site positions, without seriously disrupting homing site        binding or cleavage;    -   R51 and K98 are located in the enzyme active site and are        candidates to act as Lewis acid or to activate a proton donor in        the cleavage reaction; mutations in each of these residues have        been observed to sharply reduce I-CreI endonucleolytic activity        (R51G, K98Q);    -   five additional residues, which when mutated abolish I-CreI        endonuclease activity are located in or near the enzyme active        site (R70A, L39R, L91R, D75G, Q47H).

These studies have paved the way for a general use of meganuclease forgenome engineering. Homologous gene targeting is the most precise way tostably modify a chromosomal locus in living cells, but its lowefficiency remains a major drawback. Since meganuclease-induced DSBstimulates homologous recombination up to 10 000-fold, meganucleases aretoday the best way to improve the efficiency of gene targeting inmammalian cells (Choulika et al., 1995; Cohen-Tannoudji et al., 1998;Donoho et al., 1998; Elliott et al., 1998; Rouet et al., 1994), and tobring it to workable efficiencies in organisms such as plants (Puchta etal., 1993; Puchta et al., 1996) and insects (Rong and Golic, 2000; Rongand Golic, 2001; Rong et al., 2002).

Meganucleases have been used to induce various kinds of homologousrecombination events, such as direct repeat recombination in mammaliancells (Liang et al., 1998), plants (Siebert and Puchta, 2002), insects(Rong et al., 2002), and bacteria (Posfai et al., 1999), orinterchromosomal recombination (Moynahan and Jasin, 1997; Puchta, 1999;Richardson et al., 1998).

However, this technology is still limited by the low number of potentialnatural target sites for meganucleases: although several hundreds ofnatural homing endonucleases have been identified (Belfort and Roberts,1997; Chevalier and Stoddard, 2001), the probability to have a naturalmeganuclease cleaving a gene of interest is extremely low. The making ofartificial meganucleases with dedicated specificities would bypass thislimitation.

Artificial endonucleases with novel specificity have been made, based onthe fusion of endonucleases domains to zinc-finger DNA binding domains(Bibikova et al., 2003; Bibikova et al., 2001; Bibikova et al., 2002;Porteus and Baltimore, 2003).

Homing endonucleases have also been used as scaffolds to make novelendonucleases, either by fusion of different protein domains (Chevalieret al., 2002; Epinat et al., 2003), or by mutation of single specificamino acid residues (Seligman et al., 1997, 2002; Sussman et al., 2004;International PCT Application WO 2004/067736).

The International PCT Application WO 2004/067736 describes a generalmethod for producing a custom-made meganuclease derived from an initialmeganuclease, said meganuclease variant being able to cleave a DNAtarget sequence which is different from the recognition and cleavagesite of the initial meganuclease. This general method comprises thesteps of preparing a library of meganuclease variants having mutationsat positions contacting the DNA target sequence or interacting directlyor indirectly with said DNA target, and selecting the variants able tocleave the DNA target sequence. When the initial meganuclease is theI-CreI N75 protein a library, wherein residues 44, 68 and 70 have beenmutated was built and screened against a series of six targets close tothe I-CreI natural target site; the screened mutants have alteredbinding profiles compared to the I-CreI N75 scaffold protein; however,they cleave the I-CreI natural target site.

Seligman et al., 2002, describe mutations altering the cleavagespecificity of I-CreI. More specifically, they have studied the role ofthe nine amino acids of I-CreI predicted to directly contact the DNAtarget (Q26, K28, N30, S32, Y33, Q38, Q44, R68 and R70). Among thesenine amino acids, seven are thought to interact with nucleotides atsymmetrical positions (S32, Y33, N30, Q38, R68, Q44 and R70). Mutantshaving each of said nine amino acids and a tenth (T140) predicted toparticipate in a water-mediated interaction, converted to alanine, wereconstructed and tested in a E. coli based assay.

The resulting I-CreI mutants fell into four distinct phenotypic classesin relation to the wild-type homing site:

-   -   S32A and T140A contacts appear least important for homing site        recognition,    -   N30A, Q38A and Q44A displayed intermediate levels of activity in        each assay,    -   Q26A, R68A and Y33A are inactive,    -   K28A and R70A arc inactive and non-toxic.    -   It emerges from the results that I-CreI mutants at positions 30,        38, 44, 26, 68, 33, 28 and 70 have a modified behaviour in        relation to the wild-type I-CreI homing site.

As regards the mutations altering the seven symmetrical positions in theI-CreI homing site, it emerges from the obtained results that five ofthe seven symmetrical positions in each half-site appear to be essentialfor efficient site recognition in vivo by wild-type I-CreI: 2/21, 3/20,7/16, 8/15 and 9/14 (corresponding to positions −10/+10, −9/+9, −5/+5,−4/+4 and −3/+3 in SEQ ID NO:65). All mutants altered at these positionswere resistant to cleavage by wild-type I-CreI in vivo; however, invitro assay using E. coli appears to be more sensitive than the in vivotest and allows the detection of homing sites of wild-type I-CreI moreeffectively than the in vivo test; thus in vitro test shows that the DNAtarget of wild-type I-CreI may be the followings: gtc (recognized homingsite in all the cited documents), gcc or gtt triplet at the positions −5to −3, in reference to SEQ ID NO:65.

Seligman et al. have also studied the interaction between I-CreIposition 33 and homing site bases 2 and 21 (±10) or between I-CreIposition 32 and homing site bases 1 and 22 (±11); Y33C, Y33H, Y33R,Y33L, Y33S and Y33T mutants were found to cleave a homing site modifiedin positions ±10 that is not cleaved by I-CreI (Table 3). On the otherhand, S32K and S32R were found to cleave a homing site modified inpositions ±11 that is cleaved by I-CreI (Table 3).

Sussman et al., 2004, report studies in which the homodimeric LAGLIDADG(SEQ ID NO: 91) homing endonuclease I-CreI is altered at positions 26,and eventually 66, or at position 33, contacting the homing site basesin positions ±6 and ±10, respectively. The resulting enzymes constructs(Q26A, Q26C, Y66R, Q26C/Y66R, Y33C, Y33H) drive specific elimination ofselected DNA targets in vivo and display shifted specificities of DNAbinding and cleavage in vitro.

The overall result of the selection and characterization of enzyme pointmutants against individual target site variants is both a shift and abroadening in binding specificity and in kinetics of substrate cleavage.

Each mutant displays a higher dissociation constant (lower affinity)against the original wild-type target site than does the wild-typeenzyme, and each mutant displays a lower dissociation constant (higheraffinity) against its novel target than does the wild-type enzyme.

The enzyme mutants display similar kinetics of substrate cleavage, withshifts and broadening in substrate preferences similar to thosedescribed for binding affinities.

To reach a larger number of DNA target sequences, it would be extremelyvaluable to generate new I-CreI variants with novel specificity, ie ableto cleave DNA targets which are not cleaved by I-CreI or the fewvariants which have been isolated so far.

Such variants would be of a particular interest for genetic and genomeengineering.

SUMMARY OF THE INVENTION

Here the inventors have found mutations in positions 44, 68 and 70 ofI-CreI which result in variants able to cleave at least one homing sitemodified in positions ±3 to 5.

Therefore, the subject-matter of the present invention is a method ofpreparing a I-CreI meganuclease variant having a modified cleavagespecificity, said method comprising:

(a) replacing amino acids Q44, R68 and/or R70, in reference with I-CreIpdb accession code 1g9y, with an amino acid selected in the groupconsisting of A, D, E, G, H, K, N, P, Q, R, S, T and Y;

(b) selecting the I-CreI meganuclease variants obtained in step (a)having at least one of the following R₃ triplet cleaving profile inreference to positions −5 to −3 in a double-strand DNA target, saidpositions −5 to −3 corresponding to R₃ of the following formula I:

(SEQ ID NO: 92) 5′- R₁CAAAR₂R₃R₄R′₄R′₃R′₂TTTGR′_(l) -3′,

wherein:

R₁ is absent or present; and when present represents a nucleic acidfragment comprising 1 to 9 nucleotides corresponding either to a randomnucleic acid sequence or to a fragment of a I-CreI meganuclease homingsite situated from position −20 to −12 (from 5′ to 3′), R₁ correspondingat least to position −12 of said homing site,

R₂ represents the nucleic acid doublet ac or ct and corresponds topositions −7 to −6 of said homing site,

R₃ represents a nucleic acid triplet corresponding to said positions −5to −3, selected among g, t, c and a, except the following triplets: gtc,gcc, gtg, gtt and gct; therefore said nucleic acid triplet is preferablyselected among the following triplets: ggg, gga, ggt, ggc, gag, gaa,gat, gac, gta, gcg, gca, tgg, tga, tgt, tgc, tag, taa, tat, tac, ttg,tta, ttt, ttc, tcg, tca, tct, tcc, agg, aga, agt, agc, aag, aaa, aat,aac, atg, ata, att, atc, acg, aca, act, acc, cgg, cga, cgt, cgc, cag,caa, cat, cac, ctg, cta, ctt, ctc, ccg, cca, cct and ccc and morepreferably among the following triplets: ggg, ggt, ggc, gag, gat, gac,gta, gcg, gca, tag, taa, tat, tac, ttg, ttt, ttc, tcg, tct, tcc, agg,aag, aat, aac, att, atc, act, acc, cag, cat, cac, ctt, ctc, ccg, cct andccc,

R₄ represents the nucleic acid doublet gt or tc and corresponds topositions −2 to −1 of said homing site,

R′₁ is absent or present; and when present represents a nucleic acidfragment comprising 1 to 9 nucleotides corresponding either to a randomnucleic acid sequence or to a fragment of a I-CreI meganuclease homingsite situated from position +12 to +20 (from 5′ to 3′), R′₁corresponding at least to position +12 of said homing site,

R′₂ represents the nucleic acid doublet ag or gt, and corresponds topositions +6 to +7 of said homing site,

R′₃ represents a nucleic acid triplet corresponding to said positions +3to +5, selected among g, t, c, and a; R′₃ being different from gac, ggc,cac, aac, and agc, when R₃ and R′₃ are non-palindromic,

R′₄ represents the nucleic acid doublet ga or ac and corresponds topositions +1 to +2 of said homing site.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

-   -   Amino acid residues in a polypeptide sequence are designated        herein according to the one-letter code, in which, for example,        Q means Gln or Glutamine residue, R means Arg or Arginine        residue and D means Asp or Aspartic acid residue.    -   In the present invention, unless otherwise mentioned, the        residue numbers refer to the amino acid numbering of the I-CreI        sequence SWISSPROT P05725 or the pdb accession code 1g9y.        According to this definition, a variant named “ADR” is I-CreI        meganuclease in which amino acid residues Q44 and R68 have been        replaced by alanine and aspartic acid, respectively, while R70        has not been replaced. Other mutations that do not alter the        cleavage activity of the variant are not indicated and the        nomenclature adopted here does not limit the mutations to the        only three positions 44, 68 and 70.    -   Nucleotides are designated as follows: one-letter code is used        for designating the base of a nucleoside: a is adenine, t is        thymine, c is cytosine, and g is guanine. For the degenerated        nucleotides, r represents g or a (purine nucleotides), k        represents g or t, s represents g or c, w represents a or t, m        represents a or c, y represents t or c (pyrimidine nucleotides),        d represents g, a or t, v represents g, a or c, b represents g,        t or c, h represents a, t or c, and n represents g, a, t or c.    -   In the present application, when a sequence is given for        illustrating a recognition or homing site, it is to be        understood that it represents, from 5′ to 3′, only one strand of        the double-stranded polynucleotide.    -   The term “partially palindromic sequence”, “partially        symmetrical sequence”, “degenerate palindrome”,        “pseudopalindromic sequence” are indiscriminately used for        designating a palindromic sequence having a broken symmetry. For        example the 22 bp sequence:        c⁻¹¹a⁻¹⁰a⁻⁹a⁻⁸a⁻⁷c⁻⁶g⁻⁵t⁻⁴c⁻³g⁻²t⁻¹g₊₁a₊₂g₊₃a₊₄c₊₅a₊₆g₊₇t₊₈t₊₉t₊₁₀g₊₁₁        (SEQ ID NO: 71) is a partially palindromic sequence in which        symmetry is broken at base-pairs +/−1, 2, 6 and 7. According to        another formulation, nucleotide sequences of positions +/−8 to        11 and +/−3 to 5 are palindromic sequences. Symmetry axe is        situated between the base-pairs in positions −1 and +1. Using        another numbering, from the 5′ extremity to the 3′ extremity,        palindromic sequences are in positions 1 to 4 and 19 to 22, and        7 to 9 and 14 to 16, symmetry is broken at base-pairs 5, 6, 10,        11, 12, 13, 17 and 18, and the symmetry axe is situated between        the base-pairs in positions 11 and 12.    -   As used herein, the term “wild-type I-CreI” designates a I-CreI        meganuclase having the sequence SWISSPROT P05725 or pdb        accession code 1g9y.    -   The terms “recognition site”, “recognition sequence”, “target”,        “target sequence”, “DNA target”, “homing recognition site”,        “homing site”, “cleavage site” are indiscriminately used for        designating a 14 to 40 bp double-stranded, palindromic,        non-palindromic or partially palindromic polynucleotide sequence        that is recognized and cleaved by a meganuclease. These terms        refer to a distinct DNA location, preferably a chromosomal        location, at which a double stranded break (cleavage) is to be        induced by the meganuclease.

For example, the known homing recognition site of wild-type I-CreI isrepresented by the 22 bp sequence 5′-caaaacgtcgtgagacagtttg-3′ (SEQ IDNO: 71) or the 24 bp sequence 5′-tcaaaacgtcgtgagacagtttgg-3′ presentedin FIG. 2A (here named C1234, SEQ ID NO: 65; gtc in positions −5 to −3and gac in positions +3 to +5). This particular site is hereafter alsonamed “I-CreI natural target site”. From the natural target can bederived two palindromic sequences by mutation of the nucleotides inpositions +1, +2, +6, and +7 or −1, −2, −6 and −7: C1221 (SEQ ID NO: 12)and C4334 (SEQ ID NO:66), presented in FIG. 2A. Both have gtc inpositions −5 to −3 and gac in positions +3 to +5, and are cut by I-CreI,in vitro and in yeast.

-   -   The term “modified specificity” relates to a meganuclease        variant able to cleave a homing site that is not cleaved, in the        same conditions by the initial meganuclease (scaffold protein)        it is derived from; said initial or scaffold protein may be the        wild-type meganuclease or a mutant thereof.

Indeed, when using an in vivo assay in a yeast strain, the Inventorsfound that wild-type I-CreI cleaves not only homing sites wherein thepalindromic sequence in positions −5 to −3 is gtc (as in C 1234, C1221or C4334), but also gcc, gac, ggc, atc, ctc and ttc (FIG. 9 a).

The I-CreI D75N mutant (I-CreI N75) which may also be used as scaffoldprotein for making variants with novel specificity, cleaves not onlyhoming sites wherein the palindromic sequence in positions −5 to −3 isgtc, but also gcc, gtt, gtg, or get (FIGS. 8 and 9 a).

-   -   Heterodimeric form may be obtained for example by proceeding to        the fusion of the two monomers. Resulting heterodimeric        meganuclease is able to cleave at least one target site that is        not cleaved by the homodimeric form. Therefore a meganuclease        variant is still part of the invention when used in a        heteromeric form. The other monomer chosen for the formation of        the heterodimeric meganuclease may be another variant monomer,        but it may also be a wild-type monomer, for example a I-CreI        monomer or a I-DmoI monomer.

Thus, the inventors constructed a I-CreI variants library from a I-CreIscaffold protein (I-CreI D75N), each of them presenting at least onemutation in the amino acid residues in positions 44, 68 and/or 70 (pdbcode 1g9y), and each of them being able to cleave at least one targetsite not cleaved by the I-CreI scaffold protein.

In this particular approach, the mutation consists of the replacement ofat least one amino acid residue in position 44, 68, and/or 70 by anotherresidue selected in the group comprising A, D, E, G, H, K, N, P, Q, R,S, T and Y. Each mutated amino acid residue is changed independentlyfrom the other residues, and the selected amino acid residues may be thesame or may be different from the other amino acid residues in position44, 68 and/or 70. In this approach, the homing site, cleaved by theI-CreI meganuclease variant according to the invention but not cleavedby the I-CreI scaffold protein, is the same as described above andillustrated in FIG. 2, except that the triplet sequence in positions −5to −3 (corresponding to R₃ in formula I) and/or triplet sequence inpositions +3 to +5 (corresponding to R₃′ in formula I) differ from thetriplet sequence in the same positions in the homing sites cleaved bythe I-CreI scaffold protein.

Unexpectedly, the I-CreI meganuclease variants, obtainable by the methoddescribed above, i.e. with a “modified specificity” are able to cleaveat least one target that differs from the I-CreI scaffold protein targetin positions −5 to −3 and/or in positions +3 to +5. It must be notedthat said DNA target is not necessarily palindromic in positions +/−3 to5. I-CreI is active in homodimeric form, but may be active in aheterodimeric form. Therefore I-CreI variants according to the instantinvention could be active not only in a homodimeric form, but also in aheterodimeric form, and in both cases, they could recognize a targetwith either palindromic or non palindromic sequence in position +/−3 to5, provided that when the I-CreI N75 protein is used as scaffold, thetriplet in position −5 to −3 and/or +3 to +5 differs from gtc, gcc, gtg,gtt and gct, and from gac, ggc, cac, aac, and agc, respectively. Sinceeach monomer of I-CreI variant binds a half of the homing site, avariant able to cleave a plurality of targets could also cleave a targetwhich sequence in position +/−3 to 5 is not palindromic. Further, avariant could act both in a homodimeric form and in a heterodimericform. I-CreI variant could form a heterodimeric meganuclease, in whichthe other variant could be a wild-type I-CreI monomer, another wild-typemeganuclease monomer, such as I-DmoI, another I-CreI variant monomer, ora monomer of a variant from another meganuclease than I-CreI.

According to an advantageous embodiment of said method, the I-CreImeganuclease variant obtained in step (b) is selected from the groupconsisting of: A44/A68/A70, A44/A68/G70, A44/A68/H70, A44/A68/K70,A44/A68/N70, A44/A68/Q70, A44/A68/R70, A44/A68/S70, A44/A68/T70,A44/D68/H70, A44/D68/K70, A44/D68/R70, A44/G68/H70, A44/G68/K70,A44/G68/N70, A44/G68/P70, A44/G68/R70, A44/H68/A70, A44/H68/G70,A44/H68/H70, A44/H68/K70, A44/H68/N70, A44/H68/Q70, A44/H68/R70,A44/H68/S70, A44/H68/T70, A44/K68/A70, A44/K68/G70, A44/K68/H70,A44/K68/K70, A44/K68/N70, A44/K68/Q70, A44/K68/R70, A44/K68/S70,A44/K68/T70, A44/N68/A70, A44/N68/E70, A44/N68/G70, A44/N68/H70,A44/N68/K70, A44/N68/N70, A44/N68/Q70, A44/N68/R70, A44/N68/S70,A44/N68/T70, A44/Q68/A70, A44/Q68/D70, A44/Q68/G70, A44/Q68/H70,A44/Q68/N70, A44/Q68/R70, A44/Q68/S70, A44/R68/A70, A44/R68/D70,A44/R68/E70, A44/R68/G70, A44/R68/H70, A44/R68/K70, A44/R68/L70,A44/R68/N70, A44/R68/R70, A44/R68/S70, A44/R68/T70, A44/S68/A70,A44/S68/G70, A44/S68/K70, A44/S68/N70, A44/S68/Q70, A44/S68/R70,A44/S68/S70, A44/S68/T70, A44/T68/A70, A44/T68/G70, A44/T68/H70,A44/T68/K70, A44/T68/N70, A44/T68/Q70, A44/T68/R70, A44/T68/S70,A44/T68/T70, D44/D68/H70, D44/N68/S70, D44/R68/A70, D44/R68/K70,D44/R68/N70, D44/R68/Q70, D44/R68/R70, D44/R68/S70, D44/R68/T70,E44/H68/H70, E44/R68/A70, E44/R68/H70, E44/R68/N70, E44/R68/S70,E44/R68/T70, E44/S68/T70, G44/H68/K70, G44/Q68/H70, G44/R68/Q70,G44/R68/R70, G44/T68/D70, G44/T68/P70, G44/T68/R70, H44/A68/S70,H44/A68/T70, H44/R68/A70, H44/R68/D70, H44/R68/E70, H44/R68/G70,H44/R68/N70, H44/R68/R70, H44/R68/S70, H44/R68/T70, H44/S68/G70,H44/S68/S70, H44/S68/T70, H44/T68/S70, H44/T68/T70, K44/A68/A70,K44/A68/D70, K44/A68/E70, K44/A68/G70, K44/A68/H70, K44/A68/N70,K44/A68/Q70, K44/A68/S70, K44/A68/T70, K44/D68/A70, K44/D68/T70,K44/E68/G70, K44/E68/N70, K44/E68/S70, K44/G68/A70, K44/G68/G70,K44/G68/N70, K44/G68/S70, K44/G68/T70, K44/H68/D70, K44/H68/E70,K44/H68/G70, K44/H68/N70, K44/H68/S70, K44/H68/T70, K44/K68/A70,K44/K68/D70, K44/K68/H70, K44/K68/T70, K44/N68/A70, K44/N68/D70,K44/N68/E70, K44/N68/G70, K44/N68/H70, K44/N68/N70, K44/N68/Q70,K44/N68/S70, K44/N68/T70, K44/P68/H70, K44/Q68/A70, K44/Q68/D70,K44/Q68/E70, K44/Q68/S70, K44/Q68/T70, K44/R68/A70, K44/R68/D70,K44/R68/E70, K44/R68/G70, K44/R68/H70, K44/R68/N70, K44/R68/Q70,K44/R68/S70, K44/R68/T70, K44/S68/A70, K44/S68/D70, K44/S68/H70,K44/S681N70, K44/S68/S70, K44/S68/T70, K44/T68/A70, K44/T68/D70,K44/T68/E70, K44/T68/G70, K44/T68/H70, K44/T68/N70, K44/T68/Q70,K44/T68/S70, K44/T68/T70, N44/A68/H70, N44/A68/R70, N44/H68/N70,N44/H68/R70, N44/K68/G70, N44/K68/H70, N44/K68/R70, N44/K68/S70,N44/N68/R70, N44/P68/D70, N44/Q68/H70, N44/Q68/R70, N44/R68/A70,N44/R68/D70, N44/R68/E70, N44/R68/G70, N44/R68/H70, N44/R68/K70,N44/R68/N70, N44/R68/R70, N44/R68/S70, N44/R68/T70, N44/S68/G70,N44/S68/H70, N44/S68/K70, N44/S68/R70, N44/T68/H70, N44/T68/K70,N44/T68/Q70, N44/T68/R70, N44/T68/S70, P44/N68/D70, P44/T68/T70,Q44/A68/A70, Q44/A68/H70, Q44/A68/R70, Q44/G68/K70, Q44/G68/R70,Q44/K68/G70, Q44/N68/A70, Q44/N68/H70, Q44/N68/S70, Q44/P68/P70,Q44/Q68/G70, Q44/R68/A70, Q44/R68/D70, Q44/R68/E70, Q44/R68/G70,Q44/R68/H70, Q44/R68/N70, Q44/R68/Q70, Q44/R68/S70, Q44/S68/H70,Q44/S68/R70, Q44/S68/S70, Q44/T68/A70, Q44/T68/G70, Q44/T68/H70,Q44/T68/R70, R44/A68/G70, R44/A68/T70, R44/G68/T70, R44/H68/D70,R44/H68/T70, R44/N68/T70, R44/R68/A70, R44/R68/D70, R44/R68/E70,R44/R68/G70, R44/R68/N70, R44/R68/Q70, R44/R68/S70, R44/R68/T70,R44/S68/G70, R44/S68/N70, R44/S68/S70, R44/S68/T70, S44/D68/K70,S44/H68/R70, S44/R68/G70, S44/R68/N70, S44/R68/R70, S44/R68/S70,T44/A68/K70, T44/A68/R70, T44/H68/R70, T44/K68/R70, T44/N68/P70,T44/N68/R70, T44/Q68/K70, T44/Q68/R70, T44/R68/A70, T44/R68/D70,T44/R68/E70, T44/R68/G70, T44/R68/H70, T44/R68/K70, T44/R68/N70,T44/R68/Q70, T44/R68/R70, T44/R68/S70, T44/R68/T70, T44/S68/K70,T44/S68/R70, T44/T68/K70, and T44/T68/R70.

According to another advantageous embodiment of said method, the step(b) of selecting said I-CreI meganuclease variant is performed in vivoin yeast cells.

The subject-matter of the present invention is also the use of a I-CreImeganuclease variant as defined here above, i.e. obtainable by themethod as described above, in vitro or in vivo for non-therapeuticpurposes, for cleaving a double-strand nucleic acid target comprising atleast a 20-24 bp partially palindromic sequence, wherein at least thesequence in positions +/−8 to 11 is palindromic, and the nucleotidetriplet in positions −5 to −3 and/or the nucleotide triplet in positions+3 to +5 differs from gtc, gcc, gtg, gtt, and gct, and from gac, ggc,cac, aac and agc, respectively. Formula I describes such a DNA target.

According to an advantageous embodiment of said use, said I-CreImeganuclease variant is selected from the group consisting of:A44/A68/A70, A44/A68/G70, A44/A68/H70, A44/A68/K70, A44/A68/N70,A44/A68/Q70, A44/A68/R70, A44/A68/S70, A44/A68/T70, A44/D68/H70,A44/D68/K70, A44/D68/R70, A44/G68/H70, A44/G68/K70, A44/G68/N70,A44/G68/P70, A44/G68/R70, A44/H68/A70, A44/H68/G70, A44/H68/H70,A44/H68/K70, A44/H68/N70, A44/H68/Q70, A44/H68/R70, A44/H68/S70,A44/H68/T70, A44/K68/A70, A44/K68/G70, A44/K68/H70, A44/K68/K70,A44/K68/N70, A44/K68/Q70, A44/K68/R70, A44/K68/S70, A44/K68/T70,A44/N68/A70, A44/N68/E70, A44/N68/G70, A44/N68/H70, A44/N68/K70,A44/N68/N70, A44/N68/Q70, A44/N68/R70, A44/N68/S70, A44/N68/T70,A44/Q68/A70, A44/Q68/D70, A44/Q68/G70, A44/Q68/H70, A44/Q68/N70,A44/Q68/R70, A44/Q68/S70, A44/R68/A70, A44/R68/D70, A44/R68/E70,A44/R68/G70, A44/R68/H70, A44/R68/K70, A44/R68/L70, A44/R68/N70,A44/R68/R70, A44/R68/S70, A44/R68/T70, A44/S68/A70, A44/S68/G70,A44/S68/K70, A44/S68/N70, A44/S68/Q70, A44/S68/R70, A44/S68/S70,A44/S68/T70, A44/T68/A70, A44/T68/G70, A44/T68/H70, A44/T68/K70,A44/T68/N70, A44/T68/Q70, A44/T68/R70, A44/T68/S70, A44/T68/T70,D44/D68/H70, D44/N68/S70, D44/R68/A70, D44/R68/K70, D44/R68/N70,D44/R68/Q70, D44/R68/R70, D44/R68/S70, D44/R68/T70, E44/H68/H70,E44/R68/A70, E44/R68/H70, E44/R68/N70, E44/R68/S70, E44/R68/T70,E44/S68/T70, G44/H68/K70, G44/Q68/H70, G44/R68/Q70, G44/R68/R70,G44/T68/D70, G44/T68/P70, G44/T68/R70, H44/A68/S70, H44/A68/T70,H44/R68/A70, H44/R68/D70, H44/R68/E70, H44/R68/G70, H44/R68/N70,H44/R68/R70, H44/R68/S70, H44/R68/T70, H44/S68/G70, 1-144/S68/S70,H44/S68/T70, H44/T68/S70, H44/T68/T70, K44/A68/A70, K44/A68/D70,K44/A68/E70, K44/A68/G70, K44/A68/H70, K44/A68/N70, K44/A68/Q70,K44/A68/S70, K44/A68/T70, K44/D68/A70, K44/D68/T70, K44/E68/G70,K44/E68/N70, K44/E68/S70, K44/G68/A70, K44/G68/G70, K44/G68/N70,K44/G68/S70, K44/G68/T70, K44/H68/D70, K44/H68/E70, K44/H68/G70,K44/H68/N70, K44/H68/S70, K44/H68/T70, K44/K68/A70, K44/K68/D70,K44/K68/H70, K44/K68/T70, K44/N68/A70, K44/N68/D70, K44/N68/E70,K44/N68/G70, K44/N68/H70, K44/N68/N70, K44/N68/Q70, K44/N68/S70,K44/N68/T70, K44/P68/H70, K44/Q68/A70, K44/Q68/D70, K44/Q68/E70,K44/Q68/S70, K44/Q68/T70, K44/R68/A70, K44/R68/D70, K44/R68/E70,K44/R68/G70, K44/R68/H70, K44/R68/N70, K44/R68/Q70, K44/R68/S70,K44/R68/T70, K44/S68/A70, K44/S68/D70, K44/S68/H70, K44/S68/N70,K44/S68/S70, K44/S68/T70, K44/T68/A70, K44/T68/D70, K44/T68/E70,K44/T68/G70, K44/T68/H70, K44/T68/N70, K44/T68/Q70, K44/T68/S70,K44/T68/T70, N44/A68/H70, N44/A68/R70, N44/H68/N70, N44/H68/R70,N44/K68/G70, N44/K68/H70, N44/K68/R70, N44/K68/S70, N44/N68/R70,N44/P68/D70, N44/Q68/H70, N44/Q68/R70, N44/R68/A70, N44/R68/D70,N44/R68/E70, N44/R68/G70, N44/R68/H70, N44/R68/K70, N44/R68/N70,N44/R68/R70, N44/R68/S70, N44/R68/T70, N44/S68/G70, N44/S68/H70,N44/S68/K70, N44/S68/R70, N44/T68/H70, N44/T68/K70, N44/T68/Q70,N44/T68/R70, N44/T68/S70, P44/N68/D70, P44/T68/T70, Q44/A68/A70,Q44/A68/H70, Q44/A68/R70, Q44/G68/K70, Q44/G68/R70, Q44/K68/G70,Q44/N68/A70, Q44/N68/H70, Q44/N68/S70, Q44/P68/P70, Q44/Q68/G70,Q44/R68/A70, Q44/R68/D70, Q44/R68/E70, Q44/R68/G70, Q44/R68/H70,Q44/R68/N70, Q44/R68/Q70, Q44/R68/S70, Q44/S68/H70, Q44/S68/R70,Q44/S68/S70, Q44/T68/A70, Q44/T68/G70, Q44/T68/H70, Q44/T68/R70,R44/A68/G70, R44/A68/T70, R44/G68/T70, R44/H68/D70, R44/H68/T70,R44/N68/T70, R44/R68/A70, R44/R68/D70, R44/R68/E70, R44/R68/G70,R44/R68/N70, R44/R68/Q70, R44/R68/S70, R44/R68/T70, R44/S68/G70,R44/S68/N70, R44/S68/S70, R44/S68/T70, S44/D68/K70, S44/H68/R70,S44/R68/G70, S44/R68/N70, S44/R68/R70, S44/R68/S70, T44/A68/K70,T44/A68/R70, T44/H68/R70, T44/K68/R70, T44/N68/P70, T44/N68/R70,T44/Q68/K70, T44/Q68/R70, T44/R68/A70, T44/R68/D70, T44/R68/E70,T44/R68/G70, T44/R68/H70, T44/R68/K70, T44/R68/N70, T44/R68/Q70,T44/R68/R70, T44/R68/S70, T44/R68/T70, T44/S68/K70, T44/S68/R70,T44/T68/K70, and T44/T68/R70.

According to another advantageous embodiment of said use, the I-CreImeganuclease variant is a homodimer.

According to another advantageous embodiment of said use, said I-CreImeganuclease variant is a heterodimer.

Said heterodimer may be either a single-chain chimeric moleculeconsisting of the fusion of two different I-CreI variants as defined inthe present invention or of I-CreI scaffold protein with a I-CreIvariant as defined in the present invention. Alternatively, saidheterodimer may consist of two separate monomers chosen from twodifferent I-CreI variants as defined in the present invention or I-CreIscaffold protein and a I-CreI variant as defined in the presentinvention.

According to said use:

-   -   either the I-CreI meganuclease variant is able to cleave a DNA        target in which sequence in positions +/−3 to 5 is palindromic,    -   or, said I-CreI meganuclease variant is able to cleave a DNA        target in which sequence in positions +/−3 to 5 is        non-palindromic.

According to another advantageous embodiment of said use the cleavednucleic acid target is a DNA target in which palindromic sequences inpositions −11 to −8 and +8 to +11 are caaa and tttg, respectively.

According to another advantageous embodiment of said use, said I-CreImeganuclease variant further comprises a mutation in position 75,preferably a mutation in an uncharged amino acid, more preferably anasparagine or a valine (D75N or D75V).

According to yet another advantageous embodiment of said use, saidI-CreI meganuclease variant has an alanine (A) or an asparagine (N) inposition 44, for cleaving a DNA target comprising nucleotide a inposition −4, and/or t in position +4.

According to yet another advantageous embodiment of said use, saidI-CreI meganuclease variant has a glutamine (Q) in position 44, forcleaving a DNA target comprising nucleotide t in position −4 or a inposition +4.

According to yet another advantageous embodiment of said use, saidI-CreI meganuclease variant has a lysine (K) in position 44, forcleaving a target comprising nucleotide c in position −4, and/or g inposition +4.

The subject-matter of the present invention is also I-CreI meganucleasevariants:

-   -   Obtainable by the method of preparation as defined above;    -   Having one mutation of at least one of the amino acid residues        in positions 44, 68 and 70 of I-CreI; said mutations may be the        only ones within the amino acids contacting directly the DNA        target; and    -   Having a modified cleavage specificity in positions ±3 to 5.

Such novel I-CreI meganucleases may be used either as very specificendonucleases in in vitro digestion, for restriction or mapping use,either in vivo or ex vivo as tools for genome engineering. In addition,each one can be used as a new scaffold for a second round of mutagenesisand selection/screening, for the purpose of making novel, secondgeneration homing endonucleases.

The I-CreI meganuclease variants according to the invention are mutatedonly at positions 44, 68 and/or 70 of the DNA binding domain. However,the instant invention also includes different proteins able to formheterodimers: heterodimerization of two different proteins from theabove list result also in cleavage of non palindromic sequences, made oftwo halves from the sites cleaved by the parental proteins alone. Thiscan be obtained in vitro by adding the two different I-CreI variants inthe reaction buffer, and in vivo or ex vivo by coexpression. Anotherpossibility is to build a single-chain molecule, as described by Epinatet al. (Epinat et al., 2003). This single chain molecule would be thefusion of two different I-CreI variants, and should also result in thecleavage of chimeric, non-palindromic sequences.

According to an advantageous embodiment of said I-CreI meganucleasevariant, the amino acid residue chosen for the replacement of the aminoacid in positions 44, 68 and/or 70 is selected in the group comprisingA, D, E, G, H, K, N, P, Q, R, S, T and Y.

According to another advantageous embodiment, said I-CreI meganucleasevariant is selected in the group consisting of: A44/A68/A70,A44/A68/G70, A44/A68/H70, A44/A68/K70, A44/A68/N70, A44/A68/Q70,A44/A68/S70, A44/A68/T70, A44/D68/H70, A44/D68/K70, A44/D68/R70,A44/G68/H70, A44/G68/K70, A44/G68/N70, A44/G68/P70, A44/H68/A70,A44/H68/G70, A44/H68/H70, A44/H68/K70, A44/H68/N70, A44/H68/Q70,A44/H68/S70, A44/H68/T70, A44/K68/A70, A44/K68/G70, A44/K68/H70,A44/K68/N70, A44/K68/Q70, A44/K68/R70, A44/K68/S70, A44/K68/T70,A44/N68/A70, A44/N68/E70, A44/N68/G70, A44/N68/H70, A44/N68/K70,A44/N68/N70, A44/N68/Q70, A44/N68/R70, A44/N68/S70, A44/N68/T70,A44/Q68/A70, A44/Q68/D70, A44/Q68/G70, A44/Q68/H70, A44/Q68/N70,A44/Q68/S70, A44/R68/E70, A44/R68/K70, A44/R68/L70, A44/S68/A70,A44/S68/G70, A44/S68/N70, A44/S68/Q70, A44/S68/R70, A44/S68/S70,A44/S68/T70, A44/T68/A70, A44/T68/G70, A44/T68/H70, A44/T68/N70,A44/T68/Q70, A44/T68/S70, A44/T68/T70, D44/D68/H70, D44/N68/S70,D44/R68/A70, D44/R68/N70, D44/R68/Q70, D44/R68/R70, D44/R68/S70,D44/R68/T70, E44/H68/H70, E44/R68/A70, E44/R68/H70, E44/R68/N70,E44/R68/S70, E44/R68/T70, E44/S68/T70, G44/H68/K70, G44/Q68/H70,G44/R68/Q70, G44/T68/D70, G44/T68/P70, G44/T68/R70, H44/A68/S70,H44/A68/T70, H44/R68/D70, H44/R68/E70, H44/R68/G70, H44/R68/N70,H44/R68/R70, H44/R68/S70, H44/S68/G70, H44/S68/S70, H44/S68/T70,H44/T68/S70, H44/T68/T70, K44/A68/A70, K44/A68/D70, K44/A68/E70,K44/A68/G70, K44/A68/H70, K44/A68/N70, K44/A68/Q70, K44/D68/A70,K44/D68/T70, K44/E68/G70, K44/E68/S70, K44/G68/A70, K44/G68/G70,K44/G68/N70, K44/G68/S70, K44/G68/T70, K44/H68/D70, K44/H68/E70,K44/H68/G70, K44/H68/N70, K44/H68/S70, K44/H68/T70, K44/K68/A70,K44/K68/D70, K44/K68/H70, K44/K68/T70, K44/N68/A70, K44/N68/D70,K44/N68/E70, K44/N68/G70, K44/N68/H70, K44/N68/N70, K44/N68/Q70,K44/N68/S70, K44/N68/T70, K44/P68/H70, K44/Q68/A70, K44/Q68/D70,K44/Q68/E70, K44/Q68/S70, K44/Q68/T70, K44/R68/A70, K44/R68/D70,K44/R68/E70, K44/R68/G70, K44/R68/H70, K44/R68/N70, K44/R68/S70,K44/S68/A70, K44/S68/D70, K44/S68/H70, K44/S68/N70, K44/S68/S70,K44/S68/T70, K44/T68/A70, K44/T68/D70, K44/T68/E70, K44/T68/G70,K44/T68/H70, K44/T68/N70, K44/T68/Q70, K44/T68/S70, K44/T68/T70,N44/A68/H70, N44/H68/N70, N44/H68/R70, N44/K68/G70, N44/K68/H70,N44/K68/R70, N44/K68/S70, N44/P68/D70, N44/Q68/H70, N44/R68/A70,N44/R68/D70, N44/R68/E70, N44/R68/K70, N44/S68/G70, N44/S68/H70,N44/S68/K70, N44/S68/R70, N44/T68/H70, N44/T68/K70, N44/T68/Q70,N44/T68/S70, P44/N68/D70, P44/T68/T70, Q44/G68/K70, Q44/G68/R70,Q44/K68/G70, Q44/N68/A70, Q44/N68/H70, Q44/N68/S70, Q44/P68/P70,Q44/Q68/G70, Q44/R68/D70, Q44/R68/E70, Q44/R68/G70, Q44/R68/Q70,Q44/S68/S70, Q44/T68/A70, Q44/T68/G70, Q44/T68/H70, R44/A68/G70,R44/A68/T70, R44/G68/T70, R44/H68/D70, R44/H68/T70, R44/N68/T70,R44/R68/A70, R44/R68/D70, R44/R68/E70, R44/R68/G70, R44/R68/Q70,R44/R68/S70, R44/R68/T70, R44/S68/G70, R44/S68/N70, R44/S68/S70,R44/S68/T70, S44/D68/K70, S44/R68/R70, S44/R68/S70, T44/A68/K70,T44/N68/P70, T44/N68/R70, T44/R68/E70, T44/R68/Q70, and T44/S68/K70;said I-CreI meganuclease variant is able to cleave at least one target,as defined above, that is not cleaved by the I-CreI N75 scaffoldprotein.

According to yet another advantageous embodiment, the I-CreImeganuclease variant has an alanine (A) or an asparagine (N), inposition 44, and cleaves a target comprising the nucleotide a inposition −4, and/or t in position +4, with the exclusion of the variantspresented in Table 4 and Table 5 of the International PCT Application WO2004/067736, preferably said variant has an alanine or an asparagine.

According to yet another advantageous embodiment, the I-CreImeganuclease variant has a glutamine (Q) and cleaves a target comprisingthe nucleotide t in position −4, and/or a in position +4 in position 44,with the exclusion of the variants presented in Table 3, Table 4 andTable 5 of the International PCT Application WO 2004/067736.

According to yet another advantageous embodiment, the I-CreImeganuclease variant of the invention has a lysine (K) in position 44,and cleaves a target comprising c in position −4, and/or g in position+4, with the exclusion of the variant presented Table 5 of theInternational PCT Application WO 2004/067736.

As specified hereabove, in the frame of the definition of the I-CreImeganuclease variant in the use application, said I-CreI meganucleasevariant may be a homodimer or a heterodimer. It may be able to cleave apalindromic or a non-palindromic DNA target. It may further comprise amutation in position 75, as specified hereabove.

The subject-matter of the present invention is also a polynucleotide,characterized in that it encodes a I-CreI meganuclease variant accordingto the invention.

Further, the subject-matter of the present invention is an expressioncassette comprising said polynucleotide and regulation sequences such asa promoter, and an expression vector comprising said expressioncassette. When said variant is an heterodimer consisting of twodifferent monomers, each monomer may be expressed from a single vector(dual expression vector) or from two different vectors.

The subject-matter of the present invention is also an expressionvector, as described above, further comprising a targeting DNAconstruct.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof preferred vector is an episome, i.e., a nucleic acid capable ofextra-chromosomal replication. Preferred vectors are those capable ofautonomous replication and/or expression of nucleic acids to which theyare linked. Vectors capable of directing the expression of genes towhich they are operatively linked are referred to herein as “expressionvectors A vector according to the present invention comprises, but isnot limited to, a YAC (yeast artificial chromosome), a BAC (bacterialartificial), a baculovirus vector, a phage, a phagemid, a cosmid, aviral vector, a plasmid, a RNA vector or a linear or circular DNA or RNAmolecule which may consist of chromosomal, non chromosomal,semi-synthetic or synthetic DNA. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of“plasmids” which refer generally to circular double stranded DNA loopswhich, in their vector form are not bound to the chromosome. Largenumbers of suitable vectors are known to those of skill in the art andcommercially available, such as the following bacterial vectors: pQE7O,pQE6O, pQE-9 (Qiagen), pbs, pDIO, phagescript, psiX174. pbluescript SK,pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3,pKK233-3, pDR540, pR1T5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXTI, pSG(Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (Q1Aexpress),pET (Novagen).

Viral vectors include retrovirus, adenovirus, parvovirus (e. g.adenoassociated viruses), coronavirus, negative strand RNA viruses suchas orthomyxovirus (e. g., influenza virus), rhabdovirus (e. g., rabiesand vesicular stomatitis virus), paramyxovirus (e. g. measles andSendai), positive strand RNA viruses such as picornavirus andalphavirus, and double-stranded DNA viruses including adenovirus,herpesvirus (e. g., Herpes Simplex virus types 1 and 2, Epstein-Barrvirus, cytomegalovirus), and poxvirus (e. g., vaccinia, fowlpox andcanarypox). Other viruses include Norwalk virus, togavirus, flavivirus,reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.

Vectors can comprise selectable markers, for example: neomycinphosphotransferase, histidinol dehydrogenase, dihydrofolate reductase,hygromycin phosphotransferase, herpes simplex virus thymidine kinase,adenosine deaminase, glutamine synthetase, and hypoxanthine-guaninephosphoribosyl transferase for eukaryotic cell culture; TRP1 for S.cerevisiae; tetracycline, rifampicin or ampicillin resistance in E.coli.

Preferably said vectors are expression vectors, wherein the sequencesencoding the polypeptides of the invention are placed under control ofappropriate transcriptional and translational control elements to permitproduction or synthesis of said polypeptides. Therefore, saidpolynucleotides are comprised in expression cassette(s). Moreparticularly, the vector comprises a replication origin, a promoteroperatively linked to said encoding polynucleotide, a ribosome bindingsite, an RNA-splicing site (when genomic DNA is used), a polyadenylationsite and a transcription termination site. It also can comprise anenhancer. Selection of the promoter will depend upon the cell in whichthe polypeptide is expressed.

According to an advantageous embodiment of said expression vector, saidtargeting DNA construct comprises a sequence sharing homologies with theregion surrounding the cleavage site of the I-CreI meganuclease variantof the invention.

According to another advantageous embodiment of said expression vector,said targeting DNA construct comprises:

a) sequences sharing homologies with the region surrounding the cleavagesite of the I-CreI meganuclease variant according to claim, and

b) sequences to be introduced flanked by sequence as in a).

The subject-matter of the present invention is also a cell,characterized in that it is modified by a polynucleotide as definedabove or by a vector as defined above.

The subject-matter of the present invention is also a transgenic plant,characterized in that it comprises a polynucleotide as defined above, ora vector as defined above.

The subject-matter of the present invention is also a non-humantransgenic mammal, characterized in that it comprises a polynucleotideas defined above or a vector as defined above.

The polynucleotide sequences encoding the polypeptides as defined in thepresent invention may be prepared by any method known by the man skilledin the art. For example, they are amplified from a cDNA template, bypolymerase chain reaction with specific primers. Preferably the codonsof said cDNA are chosen to favour the expression of said protein in thedesired expression system.

The recombinant vector comprising said polynucleotides may be obtainedand introduced in a host cell by the well-known recombinant DNA andgenetic engineering techniques.

The heterodimeric meganuclease of the invention is produced byexpressing the two polypeptides as defined above; preferably saidpolypeptides are co-expressed in a host cell modified by two expressionvectors, each comprising a polynucleotide fragment encoding a differentpolypeptide as defined above or by a dual expression vector comprisingboth polynucleotide fragments as defined above, under conditionssuitable for the co-expression of the polypeptides, and theheterodimeric meganuclease is recovered from the host cell culture.

The subject-matter of the present invention is further the use of aI-CreI meganuclease variant, one or two polynucleotide(s), preferablyboth included in one expression vector (dual expression vector) or eachincluded in a different expression vector, a cell, a transgenic plant, anon-human transgenic mammal, as defined above, for molecular biology,for in vivo or in vitro genetic engineering, and for in vivo or in vitrogenome engineering, for non-therapeutic purposes.

Non therapeutic purposes include for example (i) gene targeting ofspecific loci in cell packaging lines for protein production, (ii) genetargeting of specific loci in crop plants, for strain improvements andmetabolic engineering, (iii) targeted recombination for the removal ofmarkers in genetically modified crop plants, (iv) targeted recombinationfor the removal of markers in genetically modified microorganism strains(for antibiotic production for example).

According to an advantageous embodiment of said use, it is for inducinga double-strand break in a site of interest comprising a DNA targetsequence, thereby inducing a DNA recombination event, a DNA loss or celldeath.

According to the invention, said double-strand break is for: repairing aspecific sequence, modifying a specific sequence, restoring a functionalgene in place of a mutated one, attenuating or activating an endogenousgene of interest, introducing a mutation into a site of interest,introducing an exogenous gene or a part thereof, inactivating ordeleting an endogenous gene or a part thereof, translocating achromosomal arm, or leaving the DNA unrepaired and degraded.

According to another advantageous embodiment of said use, said I-CreImeganuclease variant, polynucleotide, vector, cell, transgenic plant ornon-human transgenic mammal are associated with a targeting DNAconstruct as defined above.

The subject-matter of the present invention is also a method of geneticengineering, characterized in that it comprises a step of double-strandnucleic acid breaking in a site of interest located on a vector,comprising a DNA target of a I-CreI meganuclease variant as definedhereabove, by contacting said vector with a I-CreI meganuclease variantas defined above, thereby inducing a homologous recombination withanother vector presenting homology with the sequence surrounding thecleavage site of said I-CreI meganuclease variant.

The subject-matter of the present invention is also a method of genomeengineering, characterized in that it comprises the following steps: 1)double-strand breaking a genomic locus comprising at least onerecognition and cleavage site of a I-CreI meganuclease variant asdefined above, by contacting said cleavage site with said I-CreImeganuclease variant; 2) maintaining said broken genomic locus underconditions appropriate for homologous recombination with a targeting DNAconstruct comprising the sequence to be introduced in said locus,flanked by sequences sharing homologies with the target locus.

The subject-matter of the present invention is also a method of genomeengineering, characterized in that it comprises the following steps: 1)double-strand breaking a genomic locus comprising at least onerecognition and cleavage site of a I-CreI meganuclease variant asdefined above, by contacting said cleavage site with said I-CreImeganuclease variant; 2) maintaining said broken genomic locus underconditions appropriate for homologous recombination with chromosomal DNAsharing homologies to regions surrounding the cleavage site.

The subject-matter of the present invention is also a compositioncharacterized in that it comprises at least one I-CreI meganucleasevariant, a polynucleotide or a vector as defined above.

In a preferred embodiment of said composition, it comprises a targetingDNA construct comprising the sequence which repairs the site of interestflanked by sequences sharing homologies with the targeted locus.

The subject-matter of the present invention is also the use of at leastone I-CreI meganuclease variant, a polynucleotide or a vector, asdefined above for the preparation of a medicament for preventing,improving or curing a genetic disease in an individual in need thereof,said medicament being administrated by any means to said individual.

The subject-matter of the present invention is also the use of at leastone I-CreI meganuclease variant, a polynucleotide or a vector as definedabove for the preparation of a medicament for preventing, improving orcuring a disease caused by an infectious agent that presents a DNAintermediate, in an individual in need thereof, said medicament beingadministrated by any means to said individual.

The subject-matter of the present invention is also the use of at leastone I-CreI meganuclease variant, a polynucleotide or a vector, asdefined above, in vitro, for inhibiting the propagation, inactivating ordeleting an infectious agent that presents a DNA intermediate, inbiological derived products or products intended for biological uses orfor disinfecting an object.

In a particular embodiment, said infectious agent is a virus.

In addition to the preceding features, the invention further comprisesother features which will emerge from the description which follows,which refers to examples illustrating the I-CreI meganuclease variantsand their uses according to the invention, as well as to the appendeddrawings in which:

FIG. 1 illustrates the rationale of the experiments. (a) Structure ofI-CreI bound to its DNA target. (b) Zoom of the structure showingresidues 44, 68, 70 chosen for randomization, D75 and interacting basepairs. (c) Design of the library and targets. The interactions of I-CreIresidues Q44, R68 an R70 with DNA targets are indicated (top). Otheramino acid residues interacting directly or indirectly with the DNAtarget are not shown. Arginine (R) residue in position 44 of a I-CreImonomer directly interacts with guanine in position −5 of the targetsequence, while glutamine (Q) residue of position 44 and Arginine (R)residue of position 70 directly interact with adenine in position +4 andguanine in position +3 of the complementary strand, respectively. Thetarget described here (C1221, SEQ ID NO: 12) is a palindrome derivedfrom the I-CreI natural target (C1234, SEQ ID NO:65), and cleaved byI-CreI (Chevalier et al., 2003, precited). Cleavage positions areindicated by arrowheads. In the library, residues 44, 68 and 70 arereplaced with ADEGHKNPQRST. Since I-CreI is an homodimer, the librarywas screened with palindromic targets. Sixty four palindromic targetsresulting from substitutions in positions ±3, ±4 and ±5 were generated.A few examples of such targets are shown (bottom; SEQ ID NO: 1 to 7).

FIG. 2 illustrates the target used in the study. A. Two palindromictargets derived from the natural I-CreI target (here named C 1234, SEQID NO: 65). The I-CreI natural target contains two palindromes, boxed ingrey: the −8 to −12 and +8 to +12 nucleotides on one hand, and the −5 to−3 and +3 to +5 nucleotide on another hand. Vertical dotted line, fromwhich are numbered the nucleotide bases, represents the symmetry axe forthe palindromic sequences. From the natural target can be derived twopalindromic sequences, C1221 (SEQ ID NOS: 1-6, 81, 8-18, 82-83, 21-27,84, 29-31, 85, 33-38, 86, 40-50, 87-88, 53-59, 89, 61-63 and 90,respectively, in order of appearance) and C4334 (SEQ ID NO:66). Both arecut by I-CreI, in vitro and in yeast. Only one strand of each targetsite is shown. B. The 64 targets. The 64 targets (SEQ ID NO: 1 to 64)are derived from C1221 (SEQ ID NO: 12) a palindrome derived from theI-CreI natural target (C1234, SEQ ID NO:65), and cleaved by I-CreI(Chevalier et al., 2003, precited). They correspond to all the 24 bppalindromes resulting from substitutions at positions −5, −4, −3, +3, +4and +5.

FIG. 3 illustrates the screening of the variants. (a) Yeast aretransformed with the meganuclease expressing vector, marked with theLEU2 gene, and individually mated with yeast transformed with thereporter plasmid, marked by the TRP1 gene. In the reporter plasmid, aLacZ reporter gene is interrupted with an insert containing the site ofinterest, flanked by two direct repeats. In diploids (LEU2 TRP1),cleavage of the target site by the meganuclease (white oval) induceshomologous recombination between the two lacZ repeats, resulting in afunctional beta-galactosidase gene (grey oval), which can be monitoredby X-Gal staining. (b) Scheme of the experiment. A library of I-CreIvariants is built using PCR, cloned into a replicative yeast expressionvector and transformed in S. cerevisiae strain FYC2-6A (MATα, trp1Δ63,leu2Δ1, his3Δ200). The 64 palindromic targets are cloned in theLacZ-based yeast reporter vector, and the resulting clones transformedinto strain FYBL2-7B (MATa , ura3Δ851, trp1Δ63, leu2Δ1, lys2Δ202).Robot-assisted gridding on filter membrane is used to perform matingbetween individual clones expressing meganuclease variants andindividual clones harboring a reporter plasmid. After primary highthroughput screening, the ORF of positive clones are amplified by PCRand sequenced. 410 different variants were identified among the 2100positives, and tested at low density, to establish complete patterns,and 350 clones were validated. Also, 294 mutants were recloned in yeastvectors, and tested in a secondary screen, and results confirmed thoseobtained without recloning. Chosen clones are then assayed for cleavageactivity in a similar CHO-based assay and eventually in vitro.

FIG. 4 represents the cDNA sequence encoding the I-CreI N75 scaffoldprotein and degenerated primers used for the Ulib2 library construction.A. The coding sequence (CDS) of the scaffold protein (SEQ ID NO: 69) isfrom base-pair 1 to base-pair 501 and the “STOP” codon TGA (not shown)follows the base-pair 501. In addition to the D75N mutation, the proteinfurther contains mutations that do not alter its activity; in theprotein sequence (SEQ ID NO:70), the two first N-terminal residues aremethionine and alanine (MA), and the three C-terminal residues alanine,alanine and aspartic acid (AAD). B. Degenerated primers (SEQ ID NO: 67,68).

FIG. 5 represents the pCLS0542 meganuclease expression vector map. Themeganuclease expression vector is marked with LEU2. cDNAs encodingI-CreI meganuclease variants are cloned into this vector digested withNcoI and EagI, in order to have the variant expression driven by theinducible Gal10 promoter.

FIG. 6 represents the pCLS0042 reporter vector map. The reporter vectoris marked with TRP1 and URA3. The LacZ tandem repeats share 800 bp ofhomology, and are separated by 1,3 kb of DNA. They are surrounded by ADHpromoter and terminator sequences. Target sites are cloned into the SmaIsite.

FIG. 7 illustrates the cleavage profile of 292 I-CreI meganucleasevariants with a modified specificity. The variants derive from theI-CreI N75 scaffold protein. Proteins are defined by the amino acidpresent in positions 44, 68 and 70 (three first columns). Numeration ofthe amino acids is according to pdb accession code 1g9y. Targets aredefined by nucleotides at positions −5 to −3. For each protein, observedcleavage (1) or non observed cleavage (0) is shown for each one of the64 targets.

FIG. 8 illustrates eight examples I-CreI variants cleavage pattern. Themeganucleases are tested 4 times against the 64 targets described inFIG. 2B. The position of the different targets is indicated on the top,left panel. The variants which derive from the I-CreI N75 scaffoldprotein, are identified by the amino acids in positions 44, 68 and 70(ex: KSS is K44, S68, S70 and N75, or K44/S68/S70). Numeration of theamino acids is according to pdb code 1g9y. QRR corresponds to I-CreIN75. The cleaved targets are indicated besides the panels.

FIG. 9 illustrates the cleavage patterns of the variants. Mutants areidentified by three letters, corresponding to the residues in positions44, 68 and 70. Each mutant is tested versus the 64 targets derived fromthe I-CreI natural targets, and a series of control targets. Target mapis indicated in the top right panel. (a) Cleavage patterns in yeast(left) and mammalian cells (right) for the wild-type I-CreI (I-CreI) andI-CreI N75 (QRR) proteins, and 7 derivatives of the I-CreI N75 protein.For yeast, the initial raw data (filter) is shown. For CHO cells,quantitative raw data (ONPG measurement) are shown, values superior to0.25 are boxed, values superior to 0.5 are highlighted in medium grey,values superior to 1 in dark grey. LacZ: positive control. 0: no target.U1, U2 and U3: three different uncleaved controls. (b) Cleavage invitro. I-CreI and four mutants are tested against a set of 2 or 4targets, including the target resulting in the strongest signal in yeastand CHO. Digests are performed at 37° C. for 1 hour, with 2 nMlinearized substrate, as described in Methods. Raw data are shown forI-CreI with two different targets. With both ggg and cct, cleavage isnot detected with I-CreI.

FIG. 10 represents the statistical analysis. (a) Cleaved targets:targets cleaved by I-CreI variants are colored in grey. The number ofproteins cleaving each target is shown below, and the level of greycoloration is proportional to the average signal intensity obtained withthese cutters in yeast. (b) Analysis of 3 out of the 7 clusters. Foreach mutant cluster (clusters 1, 3 and 7), the cumulated intensities foreach target was computed and a bar plot (left column) shows indecreasing order the normalized intensities. For each cluster, thenumber of amino acid of each type at each position (44, 68 and 70) isshown as a coded histogram in the right column. The legend of amino-acidcolor code is at the bottom of the figure. (c-1 and c-2) Hierarchicalclustering of mutant and target data in yeast. Both mutants and targetswere clustered using hierarchical clustering with Euclidean distance andWard's method (Ward, J. H., American statist. Assoc., 1963, 58,236-244). Clustering was done with hclust from the R package. Mutantsand targets dendrograms were reordered to optimize positions of theclusters and the mutant dendrogram was cut at the height of 8 withdeduced clusters. QRR mutant and GTC target are indicated by an arrow.Gray levels reflects the intensity of the signal.

FIG. 11 illustrates an example of hybrid or chimeric site: gtt (SEQ IDNO: 79) and cct (SEQ ID NO: 77) are two palindromic sites derived fromthe I-CreI site. The gtt/cct hybrid site (SEQ ID NO: 80) displays thegtt sequence on the top strand in −5, −4, −3 and the cct sequence on thebottom strand in 5, 4, 3.

FIG. 12 illustrates the cleavage activity of the heterodimeric variants.Yeast were co-transformed with the KTG and QAN variants. Targetorganization is shown on the top panel: target with a single gtt, cct orgcc half site are in bold; targets with two such half sites, which areexpected to be cleaved by homo- and/or heterodimers, are in bold andhighlighted in grey; 0: no target. Results are shown on the three panelsbelow. Unexpected faint signals are observed only for gtc/cct andgtt/gtc, cleaved by KTG and QAN, respectively.

FIG. 13 represents the quantitative analysis of the cleavage activity ofthe heterodimeric variants. (a) Co-transformation of selected mutants inyeast. For clarity, only results on relevant hybrid targets are shown.The aac/acc target is always shown as an example of unrelated target.For the KTG×AGR couple, the palindromic tac and tct targets, althoughnot shown, are cleaved by AGR and KTG, respectively. Cleavage of the cattarget by the RRN mutant is very low, and could not be quantified inyeast. (b) Transient co-transfection in CHO cells. For (a) and (b),Black bars: signal for the first mutant alone; grey bars: signal for thesecond mutant alone; striped bars: signal obtained by co-expression orcotransfection.

FIG. 14 illustrates the activity of the assembled heterodimer ARS-KRE onthe selected mouse chromosome 17 DNA target. CHO-K1 cell line wereco-transfected with equimolar of target LagoZ plasmid, ARS and KREexpression plasmids, and the beta galactosidase activity was measured.Cells co-transfected with the LagoZ plasmid and the I-SceI, I-CreI, ARSor KRE recombinant plasmid or an empty plasmid were used as control.

EXAMPLES

The following examples are presented here only for illustrating theinvention and not for limiting the scope thereof. Other variants,obtained from a cDNA, which sequence differs from SEQ ID NO: 69, andusing appropriate primers, are still part of the invention.

Example 1 Screening for New Functional Endonucleases

The method for producing meganuclease variants and the assays based oncleavage-induced recombination in mammal or yeast cells, which are usedfor screening variants with altered specificity, are described in theInternational PCT Application WO 2004/067736. These assays result in afunctional LacZ reporter gene which can be monitored by standard methods(FIG. 3 a).

A) Material and Methods

a) Construction of Mutant Libraries

I-CreI wt and I-CreI D75N (or I-CreI N75) open reading frames (SEQ IDNO:69, FIG. 4A) were synthesized, as described previously (Epinat etal., N.A.R., 2003, 31, 2952-2962). Mutation D75N was introduced byreplacing codon 75 with aac. The diversity of the meganuclease librarywas generated by PCR using degenerate primers from Sigma harboring codonVVK (18 codons, amino acids ADEGHKNPQRST) at position 44, 68 and 70which interact directly with the bases at positions 3 to 5, and as DNAtemplate, the I-CreI D75N gene. Such primers allow mutation of residues44, 68 and 70 with a theoretical diversity of 12. Briefly, forwardprimer (5′-gtttaaacatcagctaagettgacctttvvkgtgacttcaaaagacccag-3′, SEQ IDNO: 67) and reverse primer(5′-gatgtagttggaaacggatccmbbatembbtacgtaaccaacgcc-3′, SEQ ID NO: 68)were used to amplify a PCR fragment in 50 μl PCR reactions: PCR productswere pooled, EtOH precipitated and resuspended in 50 μl 10 mM Tris. PCRproducts were cloned into a pET expression vector containing the I-CreID75N gene, digested with appropriate restriction enzymes. Digestion ofvector and insert DNA were conducted in two steps (single enzymedigestion) between which the DNA sample was extracted (using classicphenol:chloroform:isoamylalcohol-based methods) and EtOH-precipitated.10 μg of digested vector DNA were used for ligation, with a 5:1 excessof insert DNA. E coli TG1 cells were transformed with the resultingvector by electroporation. To produce a number of cell clones above thetheoretical diversity of the library, 6×10⁴ clones were produced.Bacterial clones were scraped from plates and the corresponding plasmidvectors were extracted and purified.

The library was recloned in the yeast pCLS0542 vector (FIG. 5), bysub-cloning a NcoI-EagI DNA fragment containing the entire I-CreI D75NORF. In this 2 micron-based replicative vector marked with the LEU2gene, I-CreI variants are under the control of a galactose induciblepromoter (Epinat et al., precited). After electroporation in E. coli,7×10⁴ clones were obtained 7×10⁴ clones, representing 12 times thetheoretical diversity at the DNA level (18³=5832). DNA was extracted andtransformed into S. cerevisiae strain FYC2-6A (MATα, trp1Δ63, leu2Δ1,his3Δ200). 13824 colonies were picked using a colony picker (QpixII,GENETIX), and grown in 144 microtiter plates.

b) Construction of Target Clones

The C1221 twenty-four by palindrome (tcaaaacgtcgtacgacgttttga, SEQ IDNO: 12) is a repeat of the half-site of the nearly palindromic naturalI-CreI target (tcaaaacgtcgtgagacagtttgg, SEQ ID NO: 65). C1221 iscleaved as efficiently as the I-CreI natural target in vitro and ex vivoin both yeast and mammalian cells. The 64 palindromic targets werederived as follows: 64 pair of oligonucleotides(ggcatacaagtttcaaaacnnngtacnnngttttgacaatcgtctgtca (SEQ ID NO: 72) andreverse complementary sequences) corresponding to the two strands of the64 DNA targets, with 12 pb of non palindromic extra sequence on eachside, were ordered form Sigma, annealed and cloned into pGEM-T Easy(PROMEGA). Next, a 400 bp PvuII fragment was excised from each one ofthe 64 pGEM-T-derived vector and cloned into the yeast vectorpFL39-ADH-LACURAZ, described previously (Epinat et al., precited), alsocalled pCLS0042 (FIG. 6), resulting in 64 yeast reporter vectors. Stepsof excision, digestion and ligation are performed using typical methodsknown by those skilled in the art. Insertion of the target sequence ismade at the SmaI site of pCLS0042. The 64 palindromic targets aredescribed in FIG. 2B (positions −5 to −3 and +3 to +5, SEQ ID NOS: 1-6,81, 8-18, 82-83, 21-27, 84, 29-31, 85, 33-38, 86, 40-50, 87-88, 53-59,89, 61-63 and 90, respectively, in order of appearance).

c) Yeast Strains and Transformation

The library of meganuclease expression variants and the A44/R68/L70variant, were transformed into strain FYC2-6A (MATα, trp1Δ63, leu2Δ1,his3Δ200).

The target plasmids were transformed into yeast strain FYBL2-7B: (MATa ,ura3Δ851, trp1Δ63, leu2Δ1, lys2Δ202).

For transformation, a classical chemical/heat choc protocol can be used,and routinely gives 10⁶ independent transformants per μg of DNA;transformants were selected on leucine drop-out synthetic medium (Gietzand Woods, 2002).

d) Mating of Meganuclease Expressing Clones and Screening in Yeast

I-CreI variant clones as well as yeast reporter strains were stocked inglycerol (20%) stock and replicated in novel microplates. Mutants weregridded on nylon filters covering YPD plates, using a high griddingdensity (about 20 spots/cm²). A second gridding process was performed onthe same filters to spot a second layer consisting of 64 or 75 differentreporter-harboring yeast strains for each variant. Briefly, eachreporter strain was spotted 13 824 times on a nylon membrane, and oneach one of this spot was spotted one out of the 13 824 yeast clonesexpressing a variant meganuclease. Membranes were placed on solid agarYPD rich medium, and incubated at 30° C. for one night, to allow mating.Next, filters were transferred to synthetic medium, lacking leucine andtryptophan, with galactose (1%) as a carbon source (and with G418 forcoexpression experiments), and incubated for five days at 37° C., toselect for diploids carrying the expression and target vectors. After 5days, filters were placed on solid agarose medium with 0.02% X-Gal in0.5 M sodium phosphate buffer, pH 7.0, 0.1% SDS, 6% dimethyl formamide(DMF), 7 mM β-mercaptoethanol, 1% agarose, and incubated at 37° C., tomonitor β-galactosidase activity. Positive clones were identified aftertwo days of incubation, according to staining. Results were analyzed byscanning and quantification was performed using a proprietary software.For secondary screening, the same procedure was followed with the 292selected positives, except that each mutant was tested 4 times on thesame membrane (see FIGS. 8 and 9 a).

d) Sequence and Re-Cloning of Primary Hits

The open reading frame (ORF) of positive clones identified during theprimary screening in yeast was amplified by PCR and sequenced. Then,ORFs were recloned using the Gateway protocol (Invitrogen). ORFs wereamplified by PCR on yeast colonies (Akada et al., Biotechniques, 28,668-670, 672-674), using primers:ggggacaagtttgtacaaaaaagcaggcttcgaaggagatagaaccatggccaataccaaatataacaaagagttcc(SEQ ID NO: 73) andggggaccactttgtacaagaaagctgggtttagtcggccgccggggaggatttcttcttctcgc (SEQ IDNO: 74) from PROLIGO. PCR products were cloned in : (i) yeast gatewayexpression vector harboring a galactose inducible promoter, LEU2 or KanRas selectable marker and a 2 micron origin of replication, and (ii) apET 24d(+) vector from NOVAGEN. Resulting clones were verified bysequencing (MILLEGEN).

B) Results

I-CreI is a dimeric homing endonuclease that cleaves a 22 bppseudo-palindromic target. Analysis of I-CreI structure bound to itsnatural target has shown that in each monomer, eight residues establishdirect interactions with seven bases (Jurica et al., 1998, precited).Residues Q44, R68, R70 contact three consecutive base pairs at position3 to 5 (and −3 to −5, FIG. 1). An exhaustive protein library vs. targetlibrary approach was undertaken to engineer locally this part of the DNAbinding interface. First, the I-CreI scaffold was mutated from D75 to Nto decrease likely energetic strains caused by the replacement of thebasic residues R68 and R70 in the library that satisfy thehydrogen-acceptor potential of the buried D75 in the I-CreI structure.Homodimers of mutant D75N (purified from E. coli cells wherein it wasover-expressed using a pET expression vector) were shown to cleave theI-CreI homing site. The D75N mutation did not affect the proteinstructure, but decreased the toxicity of I-CreI in overexpressionexperiments. Next, positions 44, 68 and 70 were randomized and 64palindromic targets resulting from substitutions in positions ±3, ±4 and±5 of a palindromic target cleaved by I-CreI (Chevalier et al., 2003,precited) were generated, as described in FIGS. 1 and 2B. Eventually,mutants in the protein library corresponded to independent combinationsof any of the 12 amino acids encoded by the vvk codon at three residuepositions. In consequence, the maximal (theoretical) diversity of theprotein library was 12³ or 1728. However, in terms of nucleic acids, thediversity is 18³ or 5832.

The resulting library was cloned in a yeast replicative expressionvector carrying a LEU2 auxotrophic marker gene and transformed into aleu2 mutant haploid yeast strain (FYC2-6A). The 64 targets were clonedin the appropriate yeast reporter vector and transformed into an haploidstrain (FYBL2-7B), resulting in 64 tester strains.

A robot-assisted mating protocol was used to screen a large number ofmeganucleases from our library. The general screening strategy isdescribed in FIG. 3 b. 13,8247 meganuclease expressing clones (about2.3-fold the theoretical diversity) were spotted at high density (20spots/cm²) on nylon filters and individually tested against each one ofthe 64 target strains (884,608 spots). 2100 clones showing an activityagainst at least one target were isolated (FIG. 3 b) and the ORFencoding the meganuclease was amplified by PCR and sequenced. 410different sequences were identified and a similar number ofcorresponding clones were chosen for further analysis. The spottingdensity was reduced to 4 spots/cm² and each clone was tested against the64 reporter strains in quadruplicate, thereby creating complete profiles(as in FIGS. 8 and 9 a). 350 positives could be confirmed. Next, toavoid the possibility of strains containing more than one clone, mutantORFs were amplified by PCR, and recloned in the yeast vector. Theresulting plasmids were individually transformed back into yeast. 294such clones were obtained and tested at low density (4 spots/cm²).Differences with primary screening were observed mostly for weaksignals, with 28 weak cleavers appearing now as negatives. Only onepositive clone displayed a pattern different from what was observed inthe primary profiling.

Example 2 I-CreI Meganuclease Variants with Different Cleavage Profiles

The validated clones from example 1 showed very diverse patterns. Someof these new profiles shared some similarity with the initial scaffoldwhereas many others were totally different. Various examples ofprofiles, including wild-type I-CreI and I-CreI N75, are shown in FIGS.8 and 9 a. The overall results (only for the 292 variants with modifiedspecificity) are summarized in FIG. 7.

Homing endonucleases can usually accommodate some degeneracy in theirtarget sequences, and one of our first findings was that the originalI-CreI protein itself cleaves seven different targets in yeast. Many ofour mutants followed this rule as well, with the number of cleavedsequences ranging from 1 to 21 with an average of 5.0 sequences cleaved(standard deviation=3.6). Interestingly, in 50 mutants (14%),specificity was altered so that they cleaved exactly one target. 37(11%) cleaved 2 targets, 61 (17%) cleaved 3 targets and 58 (17%) cleaved4 targets. For 5 targets and above, percentages were lower than 10%.Altogether, 38 targets were cleaved by the mutants (FIG. 10 a). It isnoteworthy that cleavage was barely observed on targets with an A inposition ±3, and never with targets with tgn and cgn at position ±5, ±4,±3.

These results do not limit the scope of the invention, since FIG. 7 onlyshows results obtained with 292 variants (291 out of the 1728 (or 12³)I-CreI meganuclease variants obtainable in a complete library).

Example 3 Novel Meganucleases can Cleave Novel Targets While KeepingHigh Activity and Narrow Specificity

A) Material and Methods

a) Construction of Target Clones

The 64 palindromic targets were cloned into pGEM-T Easy (PROMEGA), asdescribed in example 1. Next, a 400 bp PvuII fragment was excised andcloned into the mammalian vector pcDNA3.1-LACURAZ-ΔURA, describedpreviously (Epinat et al., precited). The 75 hybrid targets sequenceswere cloned as follows: oligonucleotides were designed that containedtwo different half sites of each mutant palindrome (PROLIGO).

b) Re-Cloning of Primary Hits

The open reading frame (ORF) of positive clones identified during theprimary screening in yeast was recloned in: (i) a CHO gateway expressionvector pCDNA6.2, following the instructions of the supplier(INVITROGEN), and ii) a pET 24d(+) vector from NOVAGEN Resulting cloneswere verified by sequencing (MILLEGEN).

c) Mammalian Cells Assay

CHO-K1 cell line from the American Type Culture Collection (ATCC) wascultured in Ham'sF12K medium supplemented with 10% Fetal Bovine Serum.For transient Single Strand Annealing (SSA) assays, cells were seeded in12 well-plates at 13.10³ cells per well one day prior transfection.Cotransfection was carried out the following day with 400 ng of DNAusing the EFFECTENE transfection kit (QIAGEN). Equimolar amounts oftarget LagoZ plasmid and expression plasmid were used. The next day,medium was replaced and cells were incubated for another 72 hours.CHO-K1 cell monolayers were washed once with PBS. The cells were thenlysed with 150 μl of lysis/revelation buffer added for β-galactosidaseliquid assay (100 ml of lysis buffer (Tris-HCl 10 mM pH7.5, NaCl 150 mM,Triton X100 0.1%, BSA 0.1 mg/ml, protease inhibitors) and 900 ml ofrevelation buffer (10 ml of Mg 100× buffer (MgCl₂ 100 mM,β-mercaptoethanol 35%), 110 ml ONPG (8 mg/ml) and 780 ml of sodiumphosphate 0.1 M pH7.5), 30 minutes on ice. Beta-galactosidase activitywas assayed by measuring optical density at 415 nm. The entire processwas performed on an automated Velocity11 BioCel platform. Thebeta-galactosidase activity is calculated as relative units normalizedfor protein concentration, incubation time and transfection efficiency.

d) Protein Expression and Purification

His-tagged proteins were over-expressed in E. coli BL21 (DE3)pLysS cellsusing pET-24d (+) vectors (NOVAGEN). Induction with IPTG (0.3 mM), wasperformed at 25° C. Cells were sonicated in a solution of 50 mM SodiumPhosphate (pH 8), 300 mM sodium chloride containing protease inhibitors(Complete EDTA-free tablets, Roche) and 5% (v/v) glycerol. Cell lysateswere centrifuged at 100000 g for 60 min. His-tagged proteins were thenaffinity-purified, using 5 ml Hi-Trap chelating HP columns (AmershamBiosciences) loaded with cobalt. Several fractions were collected duringelution with a linear gradient of imidazole (up to 0.25M imidazole,followed by plateau at 0.5 M imidazole, 0.3 M NaCl and 50 mM SodiumPhosphate pH 8). Protein-rich fractions (determined by SDS-PAGE) wereapplied to the second column. The crude purified samples were taken topH 6 and applied to a 5 ml HiTrap Heparin HP column (AmershamBiosciences) equilibrated with 20 mM Sodium Phosphate pH 6.0. Boundproteins are eluted with a sodium chloride continuous gradient with 20mM sodium phosphate and 1M sodium chloride. The purified fractions weresubmitted to SDS-PAGE and concentrated (10 kDa cut-off centriprep AmiconUltra system), frozen in liquid nitrogen and stored at −80° C. Purifiedproteins were desalted using PD10 columns (Sephadex G-25M, AmershamBiosciences) in PBS or 10 mM Tris-HCl (pH 8) buffer.

e) In Vitro Cleavage Assays

pGEM plasmids with single meganuclease DNA target cut sites were firstlinearized with XmnI. Cleavage assays were performed at 37° C. in 10 mMTris-HCl (pH 8), 50 mM NaCl, 10 mM MgCl2, 1 mM DTT and 50 μg/ml BSA. 2nM was used as target substrate concentration. A dilution range between0 and 85 nM was used for each protein, in 25 μl final volume reaction.Reactions were stopped after 1 hour by addition of 5 μl of 45% glycerol,95 mM EDTA (pH 8), 1.5% (w/v) SDS, 1.5 mg/ml proteinase K and 0.048%(w/v) bromophenol blue (6× Buffer Stop) and incubated at 37° C. for 30minutes. Digests were run on agarosse electrophoresis gel, and fragmentquantified after ethidium bromide staining, to calculate the percentageof cleavage.

B) Results

Eight representative mutants (belonging to 6 different clusters, seebelow) were chosen for further characterization (FIG. 9). First, data inyeast were confirmed in mammalian cells, by using an assay based on thetransient cotransfection of a meganuclease expressing vector and atarget vector, as described in a previous report. The 8 mutant ORFs andthe 64 targets were cloned into appropriate vectors, and arobot-assisted microtiter-based protocol was used to co-transfect in CHOcells each selected variant with each one the 64 different reporterplasmids. Meganuclease-induced recombination was measured by a standard,quantitative ONPG assay that monitors the restoration of a functionalβ-galactosidase gene. Profiles were found to be qualitatively andquantitatively reproducible in five independent experiments. As shown onFIG. 9 a, strong and medium signals were nearly always observed withboth yeast and CHO cells (with the exception of ADK), thereby validatingthe relevance of the yeast HTS process. However, weak signals observedin yeast were often not detected in CHO cells, likely due to adifference in the detection level (see QRR and targets gtg, gct, andttc). Four mutants were also produced in E. coli and purified by metalaffinity chromatography. Their relative in vitro cleavage efficienciesagainst the wild-type site and their cognate sites was determined. Theextent of cleavage under standardized conditions was assessed across abroad range of concentrations for the mutants (FIG. 9 b). Similarly, theactivity of I-CreI wt on these targets, was analysed. In many case, 100%cleavage of the substrate could not be achieved, likely reflecting thefact that these proteins may have little or no turnover (Perrin et al.,EMBO J., 1993, 12, 2939-2947; Wang et al., Nucleic Acids Res., 1997, 25,3767-3776). In general, in vitro assay confirmed the data obtained inyeast and CHO cells, but surprisingly, the gtt target was efficientlycleaved by I-CreI

Specificity shifts were obvious from the profiles obtained in yeast andCHO: the I-CreI favorite gtc target was not cleaved or barely cleaved,while signals were observed with new targets. This switch of specificitywas confirmed for QAN, DRK, RAT and KTG by in vitro analysis, as shownon FIG. 9 b. In addition, these four mutants, which display variouslevels of activity in yeast and CHO (FIG. 9 a) were shown to cleave17-60% of their favorite target in vitro (FIG. 9 b), with similarkinetics to I-CreI (half of maximal cleavage by 13-25 nM). Thus,activity was largely preserved by engineering. Third, the number ofcleaved targets varied among the mutants: strong cleavers such as QRR,QAN, ARL and KTG have a spectrum of cleavage in the range of what isobserved with I-CreI (5-8 detectable signals in yeast, 3-6 in CHO).Specificity is more difficult to compare with mutants that cleaveweakly. For example, a single weak signal is observed with DRK but mightrepresent the only detectable signal resulting from the attenuation of amore complex pattern. Nevertheless, the behavior of variants that cleavestrongly shows that engineering preserves a very narrow specificity.

Example 4 Hierarchical Clustering Defines Seven I-CreI Variant Families

A) Material and Methods

Clustering was done using hclust from the R package. We usedquantitative data from the primary, low density screening. Both variantsand targets were clustered using standard hierarchical clustering withEuclidean distance and Ward's method (Ward, J. H., American Stat.Assoc., 1963, 58, 236-244). Mutants and targets dendrograms werereordered to optimize positions of the clusters and the mutantdendrogram was cut at the height of 8 to define the cluster.

B) Results

Next, hierarchical clustering was used to determine whether familiescould be identified among the numerous and diverse cleavage patterns ofthe variants. Since primary and secondary screening gave congruentresults, quantitative data from the first round of yeast low densityscreening was used for analysis, to permit a larger sample size. Bothvariants and targets were clustered using standard hierarchicalclustering with Euclidean distance and Ward's method (Ward, J. H.,precited) and seven clusters were defined (FIG. 10 b). Detailed analysisis shown for 3 of them (FIG. 10 c) and the results are summarized inTable I.

TABLE I Cluster Analysis Nucleotide Three preferred in examples targets¹position 4 preferred amino acid² cluster (FIG. 3a) sequence % cleavage(%)¹ 44 68 70  1 QAN gtt 46.2 g 0.5 Q 77 proteins gtc 18.3 a 2.0 80.5%gtg 13.6 t 82.4 (62/77) Σ = 78.1 c 15.1  2 QRR gtt 13.4 g 0 Q R  8proteins gtc 11.8 a 4.9 100.0%  100.0%  tct 11.4 t 56.9 (8/8) (8/8) Σ =36.6 c 38.2  3 ARL gat 27.9 g 2.4 A R 65 proteins tat 23.2 a 88.9 63.0%33.8% gag 15.7 t 5.7 (41/65) (22/65) Σ = 66.8 c 3.0  4 AGR gac 22.7 g0.3 A&N R R 31 proteins tac 14.5 a 91.9 51.6% & 48.4% 67.7% gat 13.4 t6.6 35.4% 15/31 21/31 Σ = 50.6 c 1.2 (16&11/31)  5 ADK gat 29.21 g 1.681 proteins DRK tat 15.4 a 73.8 gac 11.4 t 13.4 Σ = 56.05.9 c 11.2  6KTG cct 30.1 g 0 K 51 proteins RAT tct 19.6 a 4.0 62.7% tcc 13.9 t 6.3(32/51) Σ = 63.6 c 89.7  7 cct 20.8 g 0 K 37 proteins tct 19.6 a 0.291.9% tcc 15.3 t 14.4 (34/37) Σ = 55.7 c 85.4 ¹frequencies according tothe cleavage index, as described in FIG. 10c ²in each position, residuespresent in more than ⅓ of the cluster are indicated

For each cluster, a set of preferred targets could be identified on thebasis of the frequency and intensity of the signal (FIG. 10 c). Thethree preferred targets for each cluster are indicated in Table 1, withtheir cleavage frequencies. The sum of these frequencies is ameasurement of the specificity of the cluster. For example, in cluster1, the three preferred targets (gtt/c/g), account for 78.1% of theobserved cleavage, with 46.2% for gtt alone, revealing a very narrowspecificity. Actually, this cluster includes several proteins which, asQAN, which cleaves mostly gtt (FIG. 9 a). In contrast, the threepreferred targets in cluster 2 represent only 36.6% of all observedsignals. In accordance with the relatively broad and diverse patternsobserved in this cluster, QRR cleaves 5 targets (FIG. 9 a), while othercluster members' activity are not restricted to these 5 targets.

Analysis of the residues found in each cluster showed strong biases forposition 44: Q is overwhelmingly represented in clusters 1 and 2,whereas A and N are more frequent in clusters 3 and 4, and K in clusters6 and 7. Meanwhile, these biases were correlated with strong basepreferences for DNA positions ±4, with a large majority of t:a basepairs in cluster 1 and 2, a:t in clusters 3, 4 and 5, and c:g inclusters 6 and 7 (see Table I). The structure of I-CreI bound to itstarget shows that residue Q44 interacts with the bottom strand inposition −4 (and the top strand of position +4, see FIGS. 1 b and 1 c).These results suggests that this interaction is largely conserved in ourmutants, and reveals a “code”, wherein Q44 would establish contact withadenine, A44 (or less frequently N44) with thymine, and K44 withguanine. Such correlation was not observed for positions 68 and 70.

Example 5 Variants can be Assembled in Functional Heterodimers to CleaveNew DNA Target Sequences

A) Materials and Methods

The 75 hybrid targets sequences were cloned as follows: oligonucleotideswere designed that contained two different half sites of each mutantpalindrome (PROLIGO). Double-stranded target DNA, generated by PCRamplification of the single stranded oligonucleotides, was cloned usingthe Gateway protocol (INVITROGEN) into yeast and mammalian reportervectors. Yeast reporter vectors were transformed into S. cerevisiaestrain FYBL2-7B (MATα, ura3Δ851, trp1Δ63, leu2Δ1, lys2Δ202).

B) Results

Variants are homodimers capable of cleaving palindromic sites. To testwhether the list of cleavable targets could be extended by creatingheterodimers that would cleave hybrid cleavage sites (as described inFIG. 11), a subset of I-CreI variants with distinct profiles was chosenand cloned in two different yeast vectors marked by LEU2 or KAN genes.Combinations of mutants having mutations at positions 44, 68 and/or 70and N at position 75, were then co-expressed in yeast with a set ofpalindromic and non palindromic chimeric DNA targets. An example isshown on FIG. 12: co-expression of the K44, T68, G70,N75 (KTG) and Q44,A68, N70, N75 (QAN) mutants resulted in the cleavage of two chimerictargets, gtt/gcc and gtt/cct, that were not cleaved by either mutantalone. The palindromic gtt, cct and gcc targets (and other targets ofKTG and QAN) were also cleaved, likely resulting from homodimericspecies formation, but unrelated targets were not. In addition, a gtt,cct or gcc half-site was not sufficient to allow cleavage, since suchtargets were fully resistant (see ggg/gcc, gat/gcc, gcc/tac, and manyothers, on FIG. 12). Unexpected cleavage was observed only with gtc/cctand gtt/gtc, with KTG and QAN homodimers, respectively, but signalremained very weak. Thus, efficient cleavage requires the cooperativebinding of two mutant monomers. These results demonstrate a good levelof specificity for heterodimeric species.

Altogether, a total of 112 combinations of 14 different proteins weretested in yeast, and 37.5% of the combinations (42/112) revealed apositive signal on their predicted chimeric target. Quantitative dataare shown for six examples on FIG. 13 a, and for the same sixcombinations, results were confirmed in CHO cells in transientco-transfection experiments, with a subset of relevant targets (FIG. 13b). As a general rule, functional heterodimers were always obtained whenone of the two expressed proteins gave a strong signal as homodimer. Forexample, DRN and RRN, two low activity mutants, give functionalheterodimers with strong cutters such as KTG or QRR (FIGS. 13 a and 13b) whereas no cleavage of chimeric targets could be detected byco-expression of the same weak mutants

Example 6 Cleavage of a Natural DNA Target by Assembled Heterodimer

A) Materials and Methods

a) Genome Survey

A natural target potentially cleaved by a I-CreI variant, was identifiedby scanning the public databases, for genomic sequences matching thepattern caaaacnnnnnnnnnngttttg, wherein n is a, t, c, or g (SEQ ID NO:78). The natural target DNA sequence caaaactatgtagagggttag (SEQ ID NO:75) was identified in mouse chromosome 17.

This DNA sequence is potentially cleaved by a combination of two I-CreIvariants cleaving the sequences tcaaaactatgtgaatagttttga (SEQ ID NO: 76)and tcaaaaccctgtgaagggtttga (SEQ ID NO: 77), respectively.

b) Isolation of Meganuclease Variants

Variants were selected by the cleavage-induced recombination assay inyeast, as described in example 1, using the sequencetcaaaactatgtgaatagttttga (SEQ ID NO: 76) or the sequencetcaaaaccctgtgaagggttttga (SEQ ID NO: 77) as targets.

c) Construction of the Target Plasmid

Oligonucleotides were designed that contained two different half sitesof each mutant palindrome (PROLIGO). Double-stranded target DNA,generated by PCR amplification of the single stranded oligonucleotides,was cloned using the Gateway protocol (INVITROGEN) into the mammalianreporter vector pcDNA3.1-LACURAZ-ΔURA, described previously (Epinat etal., precited), to generate the target LagoZ plasmid.

d) Construction of Meganuclease Expression Vector

The open reading frames (ORFs) of the clones identified during thescreening in yeast were amplified by PCR on yeast colony and clonedindividually in the CHO expression vector pCDNA6.2 (INVITROGEN), asdescribed in example 1. I-CreI variants were expressed under the controlof the CMV promoter.

e) Mammalian Cells Assay

CHO-K1 cell line were transiently co-transfected with equimolar amountsof target LagoZ plasmid and expression plasmids, and the betagalactosidase activity was measured as described in examples 3 and 5.

B) Results

A natural DNA target, potentially cleaved by I-CreI variants wasidentified by performing a genome survey of sequences matching thepattern caaaacnnnnnnnnnngttttg (SEQ ID NO: 78). A randomly chosen DNAsequence (SEQ ID NO: 78) identified in chromosome 17 of the mouse wascloned into a reporter plasmid. This DNA target was potentially cleavedby a combination of the I-CreI variants A44,R68,S70,N75 (ARS) andK44,R68,E70,N75 (KRE).

The co-expression of these two variants in CHO cell leads to theformation of functional heterodimer protein as shown in FIG. 14. Indeedwhen the I-CreI variants were expressed individually, virtually nocleavage activity could be detected on the mouse DNA target although theKRE protein showed a residual activity. In contrast, when these twovariants were co-expressed together with the plasmid carrying thepotential target, a strong beta-galactosidase activity could bemeasured. All together these data revealed that heterodimerizationoccurred in the CHO cells and that heterodimers were functional.

These data demonstrate that heterodimers proteins created by assemblinghomodimeric variants, extend the list of natural occurring DNA targetsequences to all the potential hybrid cleavable targets resulting fromall possible combination of the variants.

Moreover, these data demonstrated that it is possible to predict the DNAsequences that can be cleaved by a combination of variant knowing theirindividual DNA target of homodimer. Furthermore, the nucleotides atpositions 1 et 2 (and −1 and −2) of the target can be different fromgtac, indicating that they play little role in DNA/protein interaction.

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The invention claimed is:
 1. A method of preparing at least one I-CreImeganuclease variant having a modified cleavage specificity, said methodcomprising: (a) replacing amino acids Q44, R68 and/or R70, in referencewith SEQ ID NO: 70, with an amino acid selected from the groupconsisting of A, D, E, G, H, K, N, P, Q, R, S, T and Y to obtain one ormore I-CreI meganuclease variants; and (b) selecting said one or moreI-CreI meganuclease variants obtained in (a) having at least one of thefollowing R₃ triplet cleaving profile in reference to positions −5 to −3in a double-strand DNA target, said positions −5 to −3 corresponding toR₃ of the following formula I: (SEQ ID NO: 92)5′-R₁CAAAR₂R₃R₄R′₄R′₃R′₂TTTGR′₁-3′,

wherein: R₁ is absent or present; and when present represents a nucleicacid fragment comprising 1 to 9 nucleotides corresponding either to arandom nucleic acid sequence or to a fragment of a I-CreI meganucleasehoming site situated from position −20 to −12 (from 5′ to 3′), R₁corresponding at least to position −12 of said homing site, R₂represents the nucleic acid doublet ac or ct and corresponds topositions −7 to −6 of said homing site, R₃ represents a nucleic acidtriplet corresponding to said positions −5 to −3, selected among g, t, cand a, except the following triplets: gtc, gcc, gtg, gtt and gct, R₄represents the nucleic acid doublet gt or tc and corresponds topositions −2 to −1 of said homing site, R′₁ is absent or present; andwhen present represents a nucleic acid fragment comprising 1 to 9nucleotides corresponding either to a random nucleic acid sequence or toa fragment of a I-CreI meganuclease homing site situated from position+12 to +20 (from 5′ to 3′), R′₁ corresponding at least to position +12of said homing site, R′₂ represents the nucleic acid doublet ag or gt,and corresponds to positions +6 to +7 of said homing site, R′₃represents a nucleic acid triplet corresponding to said positions +3 to+5, selected among g, t, c, and a; R′₃ being different from gac, ggc,cac, aac, and agc, when R₃ and R′₃ are non-palindromic, and R′₄represents the nucleic acid doublet ga or ac and corresponds topositions +1 to +2 of said homing site.
 2. The method according to claim1, wherein said nucleic acid triplet R₃ is selected among the followingtriplets: ggg, gga, ggt, ggc, gag, gaa, gat, gac, gta, gcg, gca, tgg,tga, tgt, tgc, tag, taa, tat, tac, ttg, tta, ttt, ttc, tcg, tca, tct,tcc, agg, aga, agt, agc, aag, aaa, aat, aac, atg, ata, att, atc, acg,aca, act, acc, cgg, cga, cgt, cgc, cag, caa, cat, cac, ctg, cta, ctt,ctc, ccg, cca, cct and ccc.
 3. The method according to claim 2, whereinsaid nucleic acid triplet R₃ is selected among the following triplets:ggg, ggt, ggc, gag, gat, gac, gta, gcg, gca, tag, taa, tat, tac, ttg,ttt, ttc, tcg, tct, tcc, agg, aag, aat, aac, att, atc, act, acc, cag,cat, cac, ctt, ctc, ccg, cct and ccc.
 4. The method according to claim1, wherein the at least one I-CreI meganuclease variant selected in (b)is selected from the group consisting of: A44/A68/A70, A44/A68/G70,A44/A68/H70, A44/A68/K70, A44/A68/N70, A44/A68/Q70, A44/A68/R70,A44/A68/S70, A44/A68/T70, A44/D68/H70, A44/D68/K70, A44/D68/R70,A44/G68/H70, A44/G68/K70, A44/G68/N70, A44/G68/P70, A44/G68/R70,A44/H68/A70, A44/H68/G70, A44/H68/H70, A44/H68/K70, A44/H68/N70,A44/H68/Q70, A44/H68/R70, A44/H68/S70, A44/H68/T70, A44/K68/A70,A44/K68/G70, A44/K68/H70, A44/K68/K70, A44/K68/N70, A44/K68/Q70,A44/K68/R70, A44/K68/S70, A44/K68/T70, A44/N68/A70, A44/N68/E70,A44/N68/G70, A44/N68/H70, A44/N68/K70, A44/N68/N70, A44/N68/Q70,A44/N68/R70, A44/N68/S70, A44/N68/T70, A44/Q68/A70, A44/Q68/D70,A44/Q68/G70, A44/Q68/H70, A44/Q68/N70, A44/Q68/R70, A44/Q68/S70,A44/R68/A70, A44/R68/D70, A44/R68/E70, A44/R68/G70, A44/R68/H70,A44/R68/K70, A44/R68/L70, A44/R68/N70, A44/R68/R70, A44/R68/S70,A44/R68/T70, A44/S68/A70, A44/S68/G70, A44/S68/K70, A44/S68/N70,A44/S68/Q70, A44/S68/R70, A44/S68/S70, A44/S68/T70, A44/T68/A70,A44/T68/G70, A44/T68/H70, A44/T68/K70, A44/T68/N70, A44/T681Q70,A44/T68/R70, A44/T68/S70, A44/T68/T70, D44/D68/H70, D44/N68/S70,D44/R68/A70, D44/R68/K70, D44/R68/N70, D44/R68/Q70, D44/R68/R70,D44/R68/S70, D44/R68/T70, E44/H68/H70, E44/R68/A70, E44/R68/H70,E44/R68/N70, E44/R68/S70, E44/R68/T70, E44/S68/T70, G44/H68/K70,G44/Q68/H70, G44/R68/Q70, G44/R68/R70, G44/T68/D70, G44/T68/P70,G44/T68/R70, H44/A68/S70, H44/A68/T70, H44/R68/A70, H44/R68/D70,H44/R68/E70, H44/R68/G70, H44/R68/N70, H44/R68/R70, H44/R68/S70,H44/R68/T70, H44/S68/G70, H44/S68/S70, H44/S68/T70, H44/T68/S70,H44/T68/T70, K44/A68/A70, K44/A68/D70, K44/A68/E70, K44/A68/G70,K44/A68/H70, K44/A68/N70, K44/A68/Q70, K44/A68/S70, K44/A68/T70,K44/D68/A70, K44/D68/T70, K44/E68/G70, K44/E68/N70, K44/E68/S70,K44/G68/A70, K44/G68/G70, K44/G68/N70, K44/G68/S70, K44/G68/T70,K44/H68/D70, K44/H68/E70, K44/H68/G70, K44/H68/N70, K44/H68/S70,K44/H68/T70, K44/K68/A70, K44/K68/D70, K44/K68/H70, K44/K68/T70,K44/N68/A70, K44/N68/D70, K44/N68/E70, K44/N68/G70, K44/N68/H70,K44/N68/N70, K44/N68/Q70, K44/N68/S70, K44/N68/T70, K44/P68/H70,K44/Q68/A70, K44/Q68/D70, K44/Q68/E70, K44/Q68/S70, K44/Q68/T70,K44/R68/A70, K44/R68/D70, K44/R68/E70, K44/R68/G70, K44/R68/H70,K44/R68/N70, K44/R68/Q70, K44/R68/S70, K44/R68/T70, K44/S68/A70,K44/S68/D70, K44/S68/H70, K44/S68/N70, K44/S68/S70, K44/S68/T70,K44/T68/A70, K44/T68/D70, K44/T68/E70, K44/T68/G70, K44/T68/H70,K44/T68/N70, K44/T68/Q70, K44/T68/S70, K44/T68/T70, N44/A68/H70,N44/A68/R70, N44/H68/N70, N44/H68/R70, N44/K68/G70, N44/K68/H70,N44/K68/R70, N44/K68/S70, N44/N68/R70, N44/P68/D70, N44/Q68/H70,N44/Q68/R70, N44/R68/A70, N44/R68/D70, N44/R68/E70, N44/R68/G70,N44/R68/H70, N44/R68/K70, N44/R68/N70, N44/R68/R70, N44/R68/S70,N44/R68/T70, N44/S68/G70, N44/S68/H70, N44/S68/K70, N44/S68/R70,N44/T68/H70, N44/T68/K70, N44/T68/Q70, N44/T68/R70, N44/T68/S70,P44/N68/D70, P44/T68/T70, Q44/A68/A70, Q44/A68/H70, Q44/A68/R70,Q44/G68/K70, Q44/G68/R70, Q44/K68/G70, Q44N68/A70, Q44/N68/H70,Q44/N68/S70, Q44/P68/P70, Q44/Q68/G70, Q44/R68/A70, Q44/R68/D70,Q44/R68/E70, Q44/R68/G70, Q44/R68/H70, Q44/R68/N70, Q44/R68/Q70,Q44/R68/S70, Q44/S68/H70, Q44/S68/R70, Q44/S68/S70, Q44/T68/A70,Q44/T68/G70, Q44/T68/H70, Q44/T68/R70, R44/A68/G70, R44/A68/T70,R44/G68/T70, R44/H68/D70, R44/H68/T70, R44/N68/T70, R44/R68/A70,R44/R68/D70, R44/R68/E70, R44/R68/G70, R44/R68/N70, R44/R68/Q70,R44/R68/S70, R44/R68/T70, R44/S68/G70, R44/S68/N70, R44/S68/S70,R44/S68/T70, S44/D68/K70, S44/H68/R70, S44/R68/G70, S44/R68/N70,S44/R68/R70, S44/R68/S70, T44/A68/K70, T44/A68/R70, T44/H68/R70,T44/K68/R70, T44/N68/P70, T44/N68/R70, T44/Q68/K70, T44/Q68/R70,T44/R68/A70, T44/R68/D70, T44/R68/E70, T44/R68/G70, T44/R68/H70,T44/R68/K70, T44/R68/N70, T44/R68/Q70, T44/R68/R70, T44/R68/S70,T44/R68/T70, T44/S68/K70, T44/S68/R70, T44/T68/K70, and T44/T68/R70. 5.The method according to claim 1, wherein said selecting (b) of said atleast one I-CreI meganuclease variant is performed in vivo in yeastcells.